IL-18 inhibits growth of murine orthotopic prostate carcinomas via both adaptive and innate immune mechanisms

Interleukin(IL)-18 is a pleiotrophic cytokine with functions in immune modulation, angiogenesis and bone metabolism. In this study, the potential of IL-18 as an immunotherapy for prostate cancer (PCa) was examined using the murine model of prostate carcinoma, RM1 and a bone metastatic variant RM1(BM)/B4H7-luc. RM1 and RM1(BM)/B4H7-luc cells were stably transfected to express bioactive IL-18. These cells were implanted into syngeneic immunocompetent mice, with or without an IL-18-neutralising antibody (αIL-18, SK113AE4). IL-18 significantly inhibited the growth of both subcutaneous and orthotopic RM1 tumors and the IL-18 neutralizing antibody abrogated the tumor growth-inhibition. In vivo neutralization of interferon-gamma (IFN-γ) completely eliminated the anti-tumor effects of IL-18 confirming an essential role of IFN-γ as a down-stream mediator of the anti-tumor activity of IL-18. Tumors from mice in which IL-18 and/or IFN-γ was neutralized contained significantly fewer CD4+ and CD8+ T cells than those with functional IL-18. The essential role of adaptive immunity was demonstrated as tumors grew more rapidly in RAG1−/− mice or in mice depleted of CD4+ and/or CD8+ cells than in normal mice. The tumors in RAG1−/− mice were also significantly smaller when IL-18 was present, indicating that innate immune mechanisms are involved. IL-18 also induced an increase in tumor infiltration of macrophages and neutrophils but not NK cells. In other experiments, direct injection of recombinant IL-18 into established tumors also inhibited tumor growth, which was associated with an increase in intratumoral macrophages, but not T cells. These results suggest that local IL-18 in the tumor environment can significantly potentiate anti-tumor immunity in the prostate and clearly demonstrate that this effect is mediated by innate and adaptive immune mechanisms.

Composing maternal identities : the living realities of mothers with young adult-children in twenty-first century Australia

There is an abundance of books available on the topic of motherhood and mothering; the majority of these books focus on the vulnerability of babies and young children and the motherwork such vulnerability demands. In particular they focus on what it is right to do in the interests of the child, and particularly his or her growth and development. Such a focus is consistent in Western culture with modern moral frameworks where understandings of goodness have been assimilated to dimensions of human action rather than dimensions of human being, selfhood, or specific forms of life. As Charles Taylor has observed, much modern moral philosophy has focused =on what it is right to do rather than the nature of the good life‘ (1989, 13). The master narratives of motherhood and the prevailing social discourses of intensive1 and sacrificial2 mothering exemplify this view as such narratives and discourses depict =what mothers are expected to do [and] how mothers are supposed to be‘ (Nelson 2001, 140). From such infant/child-focused accounts a canonical maternal identity can be discerned; arguably, it is a restricted one. The majority of these books fail to address questions related to what it means be a mother in particular situated, existing, living realities. For instance, ask a mother with young children what being a mother means to her and she may speak of the challenges she faces balancing paid employment and her role as a mother, or the impact of the demands being made on her time and energy. However, ask a mother with young adult-children3 what being a mother means to her and she may speak in similar tones, but she may also speak in differing tones. For example, a "mature" mother may speak of the "empty nest", the "crowded house" and/or "its revolving front door". She may speak of issues related to the vulnerability of the long term marriage, elder care, or grandparenting, or even disillusionment and disenchantment. The purpose of this research is to explore the identity challenges and prospects of some mothers with young adult-children aged between 18 and 30 years of age in twenty-first century Australia. In interpreting the identity challenges and prospects this particular cohort of mothers encounter in their ordinary, everyday living, a diverse and particular range of maternal experiences.my own included5.have been traced, along with the social and ethical meanings ascribed in them. With an understanding and appreciation of voice as the medium which connects one's inner and outer worlds, this research illuminates the plurality of voices and the multiple layers of meaning in each of these mother's particular living and existing realities. Specifically, this research addresses the narrowly constructed, canonical maternal identity through a critical exploration and reflection on stories, shared in a research context, of the living realities of a group of self-identified "mature", middle-class, Australian mothers with children aged between 18 and 30 years of age6. By appraising the broader familial, historical, social, cultural, institutional, and, importantly, moral contexts in which these mothers are situated, 'thick descriptions' (Geertz 1973, 27)7 of maternal identities, and the challenges and prospects these mothers are negotiating, are provided. In terms of its ethical orientation, the frameworks which support and frame this research reject, repudiate and contest (Nelson 2001) the reduction of ethical concerns to individual or intellectual problems or dilemmas to be solved through the application of a theory derived from reasoned thinking. In dismissing deductive and =theoretical-juridical‘8 approaches, the individualistic orientation entrenched in contemporary Western moral thinking, expressed in the notion of '"what ought I to do" when faced with a problem, issue or dilemma of practical urgency' (Isaacs & Massey 1994, 1), is simultaneously rejected, repudiated and contested (Nelson 2001). In countering such understandings, this research reorients us to the illumination and articulation of who it is good to be, for each of these mothers, in allegiance with those goods which guide and inspire her orientations towards living a good life—a life which embraces and enhances the flourishing of herself and her significant others. With an understanding and appreciation that 'mind is never free of precommitment[—t]here is no innocent eye, nor is there one that penetrates aboriginal reality' (Bruner 1987, 32), this thesis is written with the voices of other interlocutors9. These interlocutors include the voices of my research participants whom I refer to as "research interlocutors", my textual "friends" — those scholars whose work resonates strongly with my orientations—as well as the myriad other voices that speak to mothers, for mothers and about mothers, such as those found in popular and mainstream press and culture. Sometimes these voices resonate; other times dissonance may be heard. In situating this research within these complementary frameworks, this research invites readers to join with me in considering, appreciating and appraising the narrow construction of maternal identity. I seek for this engagement, like the engagements with my research interlocutors, to be 'a meeting of voices, an authentic dialogue that is inclusive of the voices of all concerned participants' (Isaacs 2001, 6). I hope that the voices in this thesis resonate with yours (although, at times, you may feel some dissonance) and that together we can draw closer to the accounting, re-counting and re-stor(y)ing of maternal identities; like concentric circles of witness, the dialogue, ...will thus be expanded rippling into corners where one might both imagine, and least expect. Possibilities, then, are vast; the future exciting (Smith 2007, 397). This research is also shaped and guided by maternal scholarship, a relatively new field of inquiry known as 'motherhood studies' (O'Reilly 2011, xvii) which has its origins within the broader terrain of feminist scholarship. As a work of maternal scholarship, this thesis draws upon and continues the tradition of examining motherhood as it is experienced 'in a social context, as embedded in a political institution: in feminist terms' (Rich 1995, ix). It values mothers, their experiences, their stories, their lives. As such, this research is oriented towards 'matricentric feminism', a particular form of feminist inquiry, politics and theory which is consistent with and receptive to feminist frameworks of care and equal rights (O‘Reilly 2011, 25). A number of complementary conceptual frameworks have been engaged in this research with the thesis presented in three parts: the pre-figurative, configurative and re-configurative. As my particular living experiences provided the initial motivation for this research, an account of the challenges I experienced as a mother with young adult-children are outlined as a Prelude to this thesis. Attention then turns to Part One – Pre-figuring Maternal Identities in which the contextual, conceptual and methodological foundations underpinning this research are explored and outlined. In Chapter One, the prevailing cultural narratives and social discourses supporting and shaping the construction of the canonical maternal identity are outlined. Next, in setting the scholarly context, the critiques — arising from feminist and maternal scholarship — of motherhood as a patriarchal institution, mothering as experience, and mothering as work, are explored. As this research engaged with participants who are embedded in particular middle-class, heterosexual, familial and cultural structures, an exploration of family life cycle theory and main stream media accounts are also incorporated. The terrain in which "mature" mothering within an Australian context is experienced is also outlined, including the notions of "empty nests" and "crowded houses", grandparenting, elder care and women's midlife transition. Chapter Two gives an account of the conceptual ontological, ethical, identity and narrative frameworks underpinning this research. In setting the context for rich interpretations, the characteristics of being human10 are outlined before attention turns to our embodiment and embeddedness in our shared human condition11. From this point, attention then turns to understanding the moral form of human living12. In appreciating the vulnerability inherent in our shared human condition, the ways in which we may experience trouble in our lives is noted. The framing of identity constitution13 as complex, multi-faceted, relationally negotiated and composed is then outlined, followed by an understanding of why narrative is a valuable interpretive tool for interpreting and understanding human experiences. This chapter concludes with an appreciation of the ethical significance of storytelling. The research methodology is then outlined in Chapter Three. The rationale underpinning the adoption of the narrative interviewing technique of in-depth interviewing is explored. In exploring these methodological frameworks, the recruitment and interview processes involved in gathering and interpreting the recorded transcripts of ten Australian mothers with young adult-children are outlined. The method of analysis known as the Listening Guide14 best complements the multi-layered, pluri-vocal nature of narrative accounting. The final section of Chapter Three outlines The Guide, with one mother's recorded transcript used to illustrate this method's step-by-step process. Having gathered an understanding and appreciation of the pluri-vocal, multi-layered nature of narrative and identity constitution, the tone of this thesis changes in Part Two . Configuring Maternal Identities. This section consists of Chapters Four and Five and seeks to find meaning in, and make sense of, the differences and commonalities across these particular accounts. Chapter Four explores the living realities of four Australian mothers with young adult-children: Poppy, Honey, Lily and Heather. In presenting a thick description of these mothers' situated realities, the frameworks.the familial, social, cultural, historical and institutional backgrounds.which have supported and shaped each mother's experiences are illuminated. Simultaneously revealed through these particular accounts are the plurality of goods focusing and moving each mother to the moral form of life, a life of meaning and purpose. The harms challenging some mothers' moral motivations are also revealed in this chapter. Specifically illustrated in Chapter Four are the unique and qualitative differences of particular maternal identity configurations. Chapter Five reveals the commonalities amongst all of the research interlocutors' accounts. This chapter contests the individualistic orientation of many contemporary accounts of motherhood which are aimed at defining or contesting what a "good" mother ought to do. By turning away from such individualistic orientations, the chapter does not seek to define 'the content of obligation' (Taylor 1989, 3) but rather seeks to illuminate and articulate a richer, deeper understanding and appreciation of maternal be-ing and be-coming - that is, who it is good to be, for each of these mothers - in allegiance with those goods that focus and inspire her moral motivations. Part Three - Re-Configuring Maternal Identities, which is comprised of Chapter Six, draws this thesis to a close. In this final chapter, the preconceptions, conditions and aspirations for this mother-centred account of the living realities of a small, local cohort of mothers are reiterated. The insights gathered from the rich, descriptive accounts are illuminated and articulated, and the chapter closes with some suggestions for future research. In a Postlude, I reflect on how this research has been a transformative learning experience in my own life.an experience in which I have been able to not only deeply understand and appreciate the challenges and disorientation I was experiencing but also to identify and reorient my stance in relation to the good. In a practical sense, by offering thick descriptions of the living realities of this cohort of "mature" mothers, this research challenges the canonical maternal identity and questions its relevance for, and effect on, "mature" mothers' identity constitution. By bringing to light the complex existing realities of these particular mothers, this research critiques the canonical maternal identity by illustrating that each mother's life and her identity constitutions are complex, relationally negotiated and composed and that motherhood is an enduring way of being. Through these illustrations, this research engages with and extends understandings of difference feminism. This research, however, not only rejects, repudiates and contests (Nelson 2001) the narrowly defined canonical maternal identity. By illuminating and articulating the goods which shape and inspire these "mature" mothers' motherwork, this research offers a matricentric account which is consistent with and respectful of the particular, situated realities—the broader familial, social, institutional, but most importantly, moral values and frameworks—in which each mother‘s life is embedded and her motherwork oriented. By understanding and appreciating the complex and multiple webs of relationships in which each mother exists, this matricentric re-stor(y)ing of maternal experiences not only understands and appreciates the unique nature of each mother‘s existing realities, it is oriented to the continuing enhancing of the shared pursuit of the good which underpins particular maternal practices and particular maternal ways of being.

Evaluation of fibroin-based scaffolds for ocular tissue reconstruction

The epithelium of the corneolimbus contains stem cells for regenerating the corneal epithelium. Diseases and injuries affecting the limbus can lead to a condition known as limbal stem cell deficiency (LSCD), which results in loss of the corneal epithelium, and subsequent chronic inflammation and scarring of the ocular surface. Advances in the treatment of LSCD have been achieved through use of cultured human limbal epithelial (HLE) grafts to restore epithelial stem cells of the ocular surface. These epithelial grafts are usually produced by the ex vivo expansion of HLE cells on human donor amniotic membrane (AM), but this is not without limitations. Although AM is the most widely accepted substratum for HLE transplantation, donor variation, risk of disease transfer, and rising costs have led to the search for alternative biomaterials to improve the surgical outcome of LSCD. Recent studies have demonstrated that Bombyx mori silk fibroin (hereafter referred to as fibroin) membranes support the growth of primary HLE cells, and thus this thesis aims to explore the possibility of using fibroin as a biomaterial for ocular surface reconstruction. Optimistically, the grafted sheets of cultured epithelium would provide a replenishing source of epithelial progenitor cells for maintaining the corneal epithelium, however, the HLE cells lose their progenitor cell characteristics once removed from their niche. More severe ocular surface injuries, which result in stromal scarring, damage the epithelial stem cell niche, which subsequently leads to poor corneal re-epithelialisation post-grafting. An ideal solution to repairing the corneal limbus would therefore be to grow and transplant HLE cells on a biomaterial that also provides a means for replacing underlying stromal cells required to better simulate the normal stem cell niche. The recent discovery of limbal mesenchymal stromal cells (L-MSC) provides a possibility for stromal repair and regeneration, and therefore, this thesis presents the use of fibroin as a possible biomaterial to support a three dimensional tissue engineered corneolimbus with both an HLE and underlying L-MSC layer. Investigation into optimal scaffold design is necessary, including adequate separation of epithelial and stromal layers, as well as direct cell-cell contact. Firstly, the attachment, morphology and phenotype of HLE cells grown on fibroin were directly compared to that observed on donor AM, the current clinical standard substrate for HLE transplantation. The production, transparency, and permeability of fibroin membranes were also evaluated in this part of the study. Results revealed that fibroin membranes could be routinely produced using a custom-made film casting table and were found to be transparent and permeable. Attachment of HLE cells to fibroin after 4 hours in serum-free medium was similar to that supported by tissue culture plastic but approximately 6-fold less than that observed on AM. While HLE cultured on AM displayed superior stratification, epithelia constructed from HLE on fibroin maintained evidence of corneal phenotype (cytokeratin pair 3/12 expression; CK3/12) and displayed a comparable number and distribution of ÄNp63+ progenitor cells to that seen in cultures grown on AM. These results confirm the suitability of membranes constructed from silk fibroin as a possible substrate for HLE cultivation. One of the most important aspects in corneolimbal tissue engineering is to consider the reconstruction of the limbal stem cell niche to help form the natural limbus in situ. MSC with similar properties to bone marrow derived-MSC (BM-MSC) have recently been grown from the limbus of the human cornea. This thesis evaluated methods for culturing L-MSC and limbal keratocytes using various serum-free media. The phenotype of resulting cultures was examined using photography, flow cytometry for CD34 (keratocyte marker), CD45 (bone marrow-derived cell marker), CD73, CD90, CD105 (collectively MSC markers), CD141 (epithelial/vascular endothelial marker), and CD271 (neuronal marker), immunocytochemistry (alpha-smooth muscle actin; á-sma), differentiation assays (osteogenesis, adipogenesis and chrondrogenesis), and co-culture experiments with HLE cells. While all techniques supported to varying degrees establishment of keratocyte and L-MSC cultures, sustained growth and serial propagation was only achieved in serum-supplemented medium or the MesenCult-XF„¥ culture system (Stem Cell Technologies). Cultures established in MesenCult-XF„¥ grew faster than those grown in serum-supplemented medium and retained a more optimal MSC phenotype. L-MSC cultivated in MesenCult-XFR were also positive for CD141, rarely expressed £\-sma, and displayed multi-potency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of L-MSC established in MesenCult-XF„¥ medium. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker £GNp63, along with the corneal differentiation marker CK3/12. Our findings conclude that MesenCult-XFR is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells. Following on from the findings of the previous two parts, silk fibroin was tested as a novel dual-layer construct containing both an epithelium and underlying stroma for corneolimbal reconstruction. In this section, the growth and phenotype of HLE cells on non-porous versus porous fibroin membranes was compared. Furthermore, the growth of L-MSC in either serum-supplemented medium or the MesenCult-XFR culture system within fibroin fibrous mats was investigated. Lastly, the co-culture of HLE and L-MSC in serum-supplemented medium on and within fibroin dual-layer constructs was also examined. HLE on porous membranes displayed a flattened and squamous monolayer; in contrast, HLE on non-porous fibroin appeared cuboidal and stratified closer in appearance to a normal corneal epithelium. Both constructs maintained CK3/12 expression and distribution of £GNp63+ progenitor cells. Dual-layer fibroin scaffolds consisting of HLE cells and L-MSC maintained a similar phenotype as on the single layers alone. Overall, the present study proposed to create a three dimensional limbal tissue substitute of HLE cells and L-MSC together, ultimately for safe and beneficial transplantation back into the human eye. The results show that HLE and L-MSC can be cultivated separately and together whilst maintaining a clinically feasible phenotype containing a majority of progenitor cells. In addition, L-MSC were able to be cultivated routinely in the MesenCult-XF® culture system while maintaining a high purity for the MSC characteristic phenotype. However, as a serum-free culture medium was not found to sustain growth of both HLE and L-MSC, the combination scaffold was created in serum-supplemented medium, indicating that further refinement of this cultured limbal scaffold is required. This thesis has also demonstrated a potential novel marker for L-MSC, and has generated knowledge which may impact on the understanding of stromal-epithelial interactions. These results support the feasibility of a dual-layer tissue engineered corneolimbus constructed from silk fibroin, and warrant further studies into the potential benefits it offers to corneolimbal tissue regeneration. Further refinement of this technology should explore the potential benefits of using epithelial-stromal co-cultures with MesenCult-XF® derived L-MSC. Subsequent investigations into the effects of long-term culture on the phenotype and behaviour of the cells in the dual-layer scaffolds are also required. While this project demonstrated the feasibility in vitro for the production of a dual-layer tissue engineered corneolimbus, further studies are required to test the efficacy of the limbal scaffold in vivo. Future in vivo studies are essential to fully understand the integration and degradation of silk fibroin biomaterials in the cornea over time. Subsequent experiments should also investigate the use of both AM and silk fibroin with epithelial and stromal cell co-cultures in an animal model of LSCD. The outcomes of this project have provided a foundation for research into corneolimbal reconstruction using biomaterials and offer a stepping stone for future studies into corneolimbal tissue engineering.