Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references. Also available online.

Over the past several decades, our understanding of the immune system has advanced considerably. With it, an appreciation for its role in a number of diseases, such as cancer and infection has significantly grown. While our increased understanding of the immunological mechanisms underlying these diseases has improved treatment, considerable morbidity and mortality from these illnesses still exists signifying the need for more effective and innovative therapies. Dendritic cell (DC) therapy has been shown to be a promising approach to induce strong immune responses for immunotherapy, and biomaterial-based strategies have been developed to target DCs in vivo to facilitate this purpose. Given the importance of calcium in DC function and activation, we hypothesized that we could develop a biomaterial-based approach to locally and specifically control calcium signaling in DCs in vivo as a novel strategy for immunotherapy. Our first sub-hypothesis was that the calcium used to crosslink alginate gels, a commonly used biomaterial, could activate DCs in vitro; our second sub-hypothesis was that calcium ionophore A23187 could be delivered from biomaterials to activate DCs in vitro; and our third sub-hypothesis was that calcium used to crosslink alginate gels and/or controlled delivery of A23187 could increase local inflammation in vivo. We found that both the calcium released from calcium alginate gels and A23187 matured DCs and enhanced TLR-induced inflammatory cytokine secretion in vitro. Although we were unable to effectively deliver A23187 in vivo, calcium alginate gels injected subcutaneously were able to upregulate a number of inflammatory cytokines and chemokines relative to barium alginate gels. Likewise, when LPS was delivered from calcium alginate gels, the inflammatory effects of LPS on surrounding tissue were enhanced compared to when it was delivered from barium alginate gels. Thus, we confirmed that the calcium crosslinker in alginate gels could activate DCs, and provided a proof-of-principle that calcium signaling could be harnessed in vivo to enhance the immune response. Not only does this work impact the future of biomaterial design, but it may also enhance our understanding of DC biology. This thesis lays the groundwork for a novel and potentially effective strategy for enhancing DC activation in vivo, and suggests that ion signaling pathways in other cell types (both immune and non-immune) could also be targeted using biomaterials. / Engineering and Applied Sciences

The field of immunology has advanced tremendously over the last 40 years, with seminal findings that have guided the development of powerful new therapies. However, the ability to induce safe and long-lasting antigen-specific tolerance has remained elusive. A therapy that could prevent the immune system from aberrantly destroying self-tissues, without impairing its capacity to eliminate dangerous pathogens, would be transformative for the treatment of autoimmune diseases. In addition, such a therapy could also greatly advance the field of organ transplantation by inducing antigen-specific tolerance to prevent graft rejection. / Engineering and Applied Sciences

<p>Ovarian cancer is the fifth leading cause of cancer-related death in the United States and the most lethal gynecologic malignancy. Patients with ovarian cancer are generally diagnosed at stage III or IV, when ascites fluid becomes a common symptom. The volume of ascites positively correlates with the extent of ovarian cancer metastasis and negatively with prognosis; however, the mechanisms explaining their effect are unknown. </p><p>We hypothesize that ascites enriches for cancer stem-like cells. Our present study demonstrates that mice injected with ID8 cells, a murine epithelial ovarian cancer line, have remarkably shortened survival, when injected together with ascites supernatant derived from tumor-bearing mice. Moreover, compared to their counterparts cultured in regular medium, ID8 cells cultured in ascites fluid, or isolated directly from ascites, show an increased expression of stem cell markers Oct4 and CD133. These cells also exhibit enhanced self-renewing ability in sphere assay, suggesting that ascites enriches for stem-like cells. </p><p>Furthermore, we demonstrate that ascites enriches for cells expressing cell surface GRP78, a stress-inducible endoplasmic reticulum chaperone which also appears on the plasma membrane (memGRP78) of aggressive cancers. MemGRP78 + cells correlate with stem cell properties of self-renewal and tumor initiation, suggesting GRP78 is a novel stem cell marker. Importantly, antibodies against the COOH terminal domain of GRP78 significantly reduce the self-renewing ability of murine and human ovarian cancer cells pre-incubated with ascites.</p><p>In conclusion, our study demonstrates that ascites enriches for stem-like cells in ovarian cancer cell lines. Furthermore, the inhibitory effect of antibodies against the COOH terminal domain of GRP78 suggests that memGRP78 is a logical therapeutic target for ovarian cancer.</p> / Dissertation

One of the most common reasons people seek primary care and emergency care is to reduce the symptoms of allergies, such as hay fever. To meet this high demand, several recent FDA-approved methods for treating seasonal and perennial allergies have been developed, including sublingual immunotherapy tablets. Furthermore, no longer must a patient endure allergy shots; this can now be delivered sublingually. Although this method has been shown to have high safety and efficacy, very few clinicians actually utilize this form of therapy. The purpose of this paper is describe the use of sublingual immunotherapy among Nurse Practitioners (NPs) and discuss barriers that may prevent its use. Nurse Practitioners working in primary care settings were surveyed regarding their use of sublingual immunotherapy. Although many nurse practitioners treat patients with allergic disease, not one participant reported using sublingual immunotherapy. This discussion outlines some of the reasons NPs are not currently utilizing this method of allergy treatment and the findings are compared with the extant literature. This paper culminates in an evidence-based algorithm to outline best practices for utilizing sublingual immunotherapy to reduce allergy symptoms.

Immunotherapy has been proposed as an alternative to conventional cancer therapies due to its reduced toxicity and ability to induce long-lasting anti-tumor immune responses. Dendritic cell (DC) vaccination is one immune-based anti-cancer strategy that has received attention due to the ability of DC to process and present antigen to T lymphocytes to initiate immune responses. However, the clinical efficacy of DC-based immunotherapy against established cancers in humans has been extremely low and despite recent advances, objective response rates in DC vaccine trials are rarely above 10%. This lack of efficacy is due in part to immunosuppressive factors, such as transforming growth factor &beta (TGF-&beta), present in the tumor microenvironment that promote tumor immune escape. Therefore, TGF-&beta represents a major barrier to effective cancer immunotherapy and strategies to neutralize this cytokine may lead to more efficacious DC vaccines.In this study, we employed two small molecule transforming growth factor &beta receptor type I (T&betaRI/ALK5) kinase inhibitors (HTS466284 and SM16) in combination with DC vaccines to treat established TGF-&beta-secreting 4T1 mammary tumors. The results demonstrate that while both inhibitors blocked the effects of TGF-&beta in vitro, HTS466284 by itself or in combination with DC vaccination was unable to consistently control the growth and metastasis of established 4T1 tumors. In contrast, SM16 inhibited the growth of established tumors when delivered orally and suppressed the formation of pulmonary metastases when delivered orally or via daily intraperitoneal (i.p.) injection. The efficacy of SM16 was dependent on cellular immunity as this drug had no effect in immunodeficient SCID mice. Furthermore, orally delivered SM16 in combination with DC vaccination led to complete tumor regression in several mice that correlated with increased T cell infiltration of the primary tumor and enhanced in vitro IFN-gamma production and tumor-specific cytolytic activity by splenocytes. Finally, a suboptimal dose of SM16 that failed to control primary tumor growth on its own synergized with DC vaccination to inhibit the growth of established 4T1 tumors. These findings suggest that blockade of TGF-&beta signaling using a small molecule T&betaRI/ALK5 kinase antagonist may be an effective strategy to bolster the efficacy of DC-based cancer vaccines.

Focused ultrasound (FUS) can temporarily increase blood-brain barrier (BBB) permeability and locally deliver therapeutic agents to the brain. To date, applications of FUS for treatment of Alzheimer’s disease (AD) have not been explored. Here, I propose that FUS can facilitate a rapid reduction in amyloid-β peptide (Aβ) pathology in a mouse model of AD. Firstly, FUS was used to enhance delivery of an antibody directed against Aβ, which aggregates and forms extracellular plaques. FUS mediated the delivery of antibodies to the targeted right cortex by 4 hours post-treatment and antibodies remained bound to Aβ plaques for 4 days. At 4 days post-treatment, stereological quantification of plaque burden demonstrated a significant reduction of 23%. Secondly, FUS treatment alone resulted in a significant reduction in plaque load (13%). I then investigated effects of FUS that may contribute to Aβ plaque reduction, specifically the delivery of endogenous antibodies to the brain and, activation of microglia and astrocytes. Endogenous immunoglobulin was found bound to plaques within the treated cortex at 4 days post-FUS. Western blot analysis confirmed that immunoglobulin levels were increased significantly. Further, FUS led to a time-dependent increase in glial response. The expression of ionized calcium-binding adaptor molecule 1, a marker of phagocytic microglia, was increased at 4 hours and 4 days, and it was resolved by 15 days. Astrocytes had a slightly delayed response, with an increase in the expression of glial fibrillary acidic protein at 4 days, which declined by 15 days. After 4 days, microglia and astrocytes had significantly greater volumes and surface areas, signifying enhanced activation in the FUS-treated cortex, without an apparent increase in cell count. Co-localization of Aβ within activated glia revealed a significant increase in Aβ internalization following FUS. In conclusion, it was demonstrated that the delivery of exogenous antibodies by FUS, and FUS alone can lead to plaque reduction. Mechanisms by which FUS alone reduces plaque load may include entry of endogenous antibodies to the brain and the induction of a transient glial response. This work details acute effects of FUS that highlight the promise of this delivery method for AD treatment.

The goal of cancer immunotherapies is to boost the immune system's ability to detect tumor antigens and mount an effective anti-tumor immune response. Currently, adoptive T cell transfer therapy (ACT), the administration of ex vivo expanded autologous tumor-specific T cells, is one of the most promising immunotherapies under development; however, its efficacy has been limited so far with a mere 10% complete remission rate in the most successful clinical trials. The prolonged ex vivo culture process is a potential reason for this ineffectiveness because the transfused cells may reach replicative senescence and immunosenescence prior to patient transfer. The objective of this thesis is to offer two approaches towards an improvement of treatment efficacy. First, we generated a 'senescence metric' from the identification of biomarkers that can be used in the clinic towards predicting age and responsiveness of ex vivo expanded T cells. The second approach is to understand at the molecular level the changes that occur during ex vivo expansion to devise improved ACT protocols. In particular, we focused on the shift towards a pro-oxidizing environment and its potential effects on calcium signaling. The combined development and application of microfluidic technologies and computational models in this thesis facilitated our investigations of the phenotypic and signaling changes occurring in T cells during the progression towards immunosenescence. Our findings of altered T cell properties over long term culture provide insight for the design of future cancer immunotherapy protocols.

Focused ultrasound (FUS) can temporarily increase blood-brain barrier (BBB) permeability and locally deliver therapeutic agents to the brain. To date, applications of FUS for treatment of Alzheimer’s disease (AD) have not been explored. Here, I propose that FUS can facilitate a rapid reduction in amyloid-β peptide (Aβ) pathology in a mouse model of AD. Firstly, FUS was used to enhance delivery of an antibody directed against Aβ, which aggregates and forms extracellular plaques. FUS mediated the delivery of antibodies to the targeted right cortex by 4 hours post-treatment and antibodies remained bound to Aβ plaques for 4 days. At 4 days post-treatment, stereological quantification of plaque burden demonstrated a significant reduction of 23%. Secondly, FUS treatment alone resulted in a significant reduction in plaque load (13%). I then investigated effects of FUS that may contribute to Aβ plaque reduction, specifically the delivery of endogenous antibodies to the brain and, activation of microglia and astrocytes. Endogenous immunoglobulin was found bound to plaques within the treated cortex at 4 days post-FUS. Western blot analysis confirmed that immunoglobulin levels were increased significantly. Further, FUS led to a time-dependent increase in glial response. The expression of ionized calcium-binding adaptor molecule 1, a marker of phagocytic microglia, was increased at 4 hours and 4 days, and it was resolved by 15 days. Astrocytes had a slightly delayed response, with an increase in the expression of glial fibrillary acidic protein at 4 days, which declined by 15 days. After 4 days, microglia and astrocytes had significantly greater volumes and surface areas, signifying enhanced activation in the FUS-treated cortex, without an apparent increase in cell count. Co-localization of Aβ within activated glia revealed a significant increase in Aβ internalization following FUS. In conclusion, it was demonstrated that the delivery of exogenous antibodies by FUS, and FUS alone can lead to plaque reduction. Mechanisms by which FUS alone reduces plaque load may include entry of endogenous antibodies to the brain and the induction of a transient glial response. This work details acute effects of FUS that highlight the promise of this delivery method for AD treatment.

Nitric oxide (NO) has been shown to be lethal for a variety of intracellular pathogens including Leishmania. In murine models, the inducible nitric oxide synthase gene (iNOS) is expressed in activated macrophages and is involved in the synthesis of NO. Because the role of NO and iNOS in human leishmaniasis was less clear, we examined the expression of the iNOS gene in human macrophages infected with Leishmania in vitro and in biopsy tissue from subjects with cutaneous leishmaniasis. / Pentavalent antimony (Sb5+) in the form of Pentostam(TM) or Glucantime(TM) is still the treatment of choice despite its toxicity. Aldara(TM) (5% imiquimod) is an immune-response modifying agent that has been approved by the Food and Drug Administration in the USA for treating genital warts caused by papillomaviruses. We conducted an open-label, prospective study of combined Glucantime(TM) + Aldara(TM) therapy in subjects with CL who had previously failed a complete course of Glucantime(TM) treatment at regular doses. (Abstract shortened by UMI.)