In vivo assessment of specific cytotoxic T lymphocyte killing

The direct killing of target cells by cytotoxic T lymphocytes (CTLs) plays a fundamental role in protective immunity to viral, bacterial, protozoan and fungi infections, as well as to tumor cells. In vivo cytotoxic assays take into account the interaction of target and effector cells in the context of the proper microenvironment making the analysis biologically more relevant than in vitro cytotoxic assays. Thus, the development, improvement and validation of in vivo methods are necessary in view of the importance of the results they may provide. We describe and discuss in this manuscript a method to evaluate in vivo specific cytotoxic T lymphocyte killing. We used as model system mice immunized with human recombinant replication-deficient adenovirus 5 (HAd5) containing different transgenes as the trigger of a CTL-mediated immune response. To these mice, we adoptively transferred syngeneic cells labeled with different vital fluorescent dyes. Donor cells were pulsed (target) or not (control non-target) with distinct CD8 T-cell epitopes, mixed in a 1:1 ratio and injected i.v. into immunized or non-immunized recipient mice. After 18-24h, spleen cells are collected and analysed by flow cytometry. A deviation from the 1:1 ratio of control and target cell populations indicates antigen specific lysis of target cells

In vitro generation and characterization of acute myeloid leukemia-reactive CD8 + cytotoxic T-lymphocyte clones from healthy donors

Donor-derived CD8+ cytotoxic T lymphocytes (CTLs) eliminating host leukemic cells mediate curative graft-versus-leukemia (GVL) reactions after allogeneic hematopoietic stem cell transplantation (HSCT). The leukemia-reactive CTLs recognize hematopoiesis-restricted or broadly expressed minor histocompatibility and leukemia-associated peptide antigens that are presented by human leukocyte antigen (HLA) class I molecules on recipient cells. The development of allogeneic CTL therapy in acute myeloid leukemia (AML) is hampered by the poor efficiency of current techniques for generating leukemia-reactive CTLs from unprimed healthy donors in vitro. In this work, a novel allogeneic mini-mixed lymphocyte/leukemia culture (mini-MLLC) approach was established by stimulating CD8+ T cells isolated from peripheral blood of healthy donors at comparably low numbers (i.e. 10e4/well) with HLA class I-matched primary AML blasts in 96-well microtiter plates. Before culture, CD8+ T cells were immunomagnetically separated into CD62L(high)+ and CD62L(low)+/neg subsets enriched for naive/central memory and effector memory cells, respectively. The application of 96-well microtiter plates aimed at creating multiple different responder-stimulator cell compositions in order to provide for the growth of leukemia-reactive CTLs optimized culture conditions by chance. The culture medium was supplemented with interleukin (IL)-7, IL-12, and IL-15. On day 14, IL-12 was replaced by IL-2. In eight different related and unrelated donor/AML pairs with complete HLA class I match, numerous CTL populations were isolated that specifically lysed myeloid leukemias in association with various HLA-A, -B, or -C alleles. These CTLs recognized neither lymphoblastoid B cell lines of donor and patient origin nor primary B cell leukemias expressing the corresponding HLA restriction element. CTLs expressed T cell receptors of single V-beta chain families, indicating their clonality. The vast majority of CTL clones were obtained from mini-MLLCs initiated with CD8+ CD62L(high)+ cells. Using antigen-specific stimulation, multiple CTL populations were amplified to 10e8-10e10 cells within six to eight weeks. The capability of mini-MLLC derived AML-reactive CTL clones to inhibit the engraftment of human primary AML blasts was investigated in the immunodeficient nonobese diabetic/severe combined immune deficient IL-2 receptor common γ-chain deficient (NOD/SCID IL2Rγnull) mouse model. The leukemic engraftment in NOD/SCID IL2Rγnull was specifically prevented if inoculated AML blasts had been pre-incubated in vitro with AML-reactive CTLs, but not with anti-melanoma control CTLs. These results demonstrate that myeloid leukemia-specific CTL clones capable of preventing AML engraftment in mice can be rapidly isolated from CD8+ CD62L(high)+ T cells of healthy donors in vitro. The efficient generation and expansion of these CTLs by the newly established mini-MLLC approach opens the door for several potential applications. First, CTLs can be used within T cell-driven antigen identification strategies to extend the panel of molecularly defined AML antigens that are recognizable by T cells of healthy donors. Second, because these CTLs can be isolated from the stem cell donor by mini-MLLC prior to transplantation, they could be infused into AML patients as a part of the stem cell allograft, or early after transplantation when the leukemia burden is low. The capability of these T cells to expand and function in vivo might require the simultaneous administration of AML-reactive CD4+ T cells generated by a similar in vitro strategy or, less complex, the co-transfer of CD8-depleted donor lymphocytes. To prepare clinical testing, the mini-MLLC approach should now be translated into a protocol that is compatible with good manufacturing practice guidelines.

Clonable progenitors committed to the T lymphocyte lineage in the mouse bone marrow; use of an extrathymic pathway

We searched for clonable committed T cell progenitors in the adult mouse bone marrow and isolated rare (≈0.05%) cells with the Thy-1hiCD2−CD16+CD44hiCD25−Lin− phenotype. In vivo experiments showed that these cells were progenitors committed only to reconstituting the T cell lineage of irradiated Ly5 congenic hosts. Reconstitution of the thymus was minimal compared with that of the bone marrow, spleen, and lymph nodes. At limiting dilutions, donor T cell reconstitution of the spleen frequently occurred without detectable donor cells in the thymus. Progenitors were capable of rapidly reconstituting athymic hosts. In conclusion, the clonable bone marrow progenitors were capable of T cell reconstitution predominantly by means of an extrathymic pathway.

Similarity not favourability : the role of donor prototypes in predicting willingness to donate organs while living

Using an extended Prototype/Willingness Model, we examined the predictors of willingness to donate an organ to a partner/family member and a stranger while living. A questionnaire assessed university students’ (N = 284) attitudes, subjective norm, prototype favourability, prototype similarity, moral norm, and willingness to donate organs in each recipient scenario. All variables, except prototype favourability, predicted willingness to donate organs in both situations. Future strategies should emphasise perceived approval from important others for living donation, the consistency of living donation with one’s own morals, and encourage perceptions of similarity between oneself and living donors to increase acceptance of living donation.

Knowing a donor and identifying as one : determinants of people's willingness for related and anonymous living donation in Australia

While LRD (living donation to a genetically/emotionally related recipient) is well established in Australia, LAD (living anonymous donation to a stranger) is rare. Given the increasing use of LAD overseas, Australia may likely follow suit. Understanding the determinants of people’s willingness for LAD is essential but infrequently studied in Australia. Consequently, we compared the determinants of people’s LRD and LAD willingness, and assessed whether these determinants differed according to type of living donation. We surveyed 487 health students about their LRD and LAD willingness, attitudes, identity, prior experience with blood and organ donation, deceased donation preference, and demographics. We used Structural Equation Modelling (SEM) to identify the determinants of willingness for LRD and LAD and paired sample t-tests to examine differences in LRD and LAD attitudes, identity, and willingness. Mean differences in willingness (LRD 5.93, LAD 3.92), attitudes (LRD 6.43, LAD 5.53), and identity (LRD 5.69, LAD 3.58) were statistically significant. Revised SEM models provided a good fit to the data (LRD: x2 (41) = 67.67, p = 0.005, CFI = 0.96, RMSEA = 0.04; LAD: x2 (40) = 79.64, p < 0.001, CFI = 0.95, RMSEA = 0.05) and explained 45% and 54% of the variation in LRD and LAD willingness, respectively. Four common determinants of LRD and LAD willingness emerged: identity, attitude, past blood donation, and knowing a deceased donor. Religious affiliation and deceased donation preference predicted LAD willingness also. Identifying similarities and differences in these determinants can inform future efforts aimed at understanding people’s LRD and LAD willingness and the evaluation of potential living donor motives. Notably, this study highlights the importance of people’s identification as a living donor as a motive underlying their willingness to donate their organs while living.

Disclosing donation decisions : the role of organ donor prototypes in an extended theory of planned behaviour

This study explored the role of donor prototype evaluations (perceptions of the typical organ donor) in organ donation communication decisions using an extended theory of planned behaviour (TPB) model. The model incorporated attitude, subjective norm, perceived behavioural control, moral norm, self-identity, and donor prototype evaluations to predict intentions to record consent on an organ donor register and discuss the organ donation decision with significant others. Participants completed surveys assessing the extended TPB constructs related to registering (n = 359) and discussing (n = 282). Results supported a role for donor prototype evaluations in predicting discussing intentions only. Both extended TPB structural equation models were a good fit to the data, accounting for 74 and 76% of the variance in registering and discussing intentions, respectively. Participants’ self-reported discussing behaviour (but not registering behaviour given low numbers of behavioural performers) was assessed 4 weeks later, with discussing intention as the only significant predictor of behaviour (Nagelkerke R2 = 0.11). These findings highlight the impact of people's perceptions of a typical donor on their decisions to discuss their organ donation preference, assisting our understanding of the factors influencing individuals' communication processes in efforts to bridge the gap between organ supply and demand.

Novel ruthenium bipyridyl dyes with s-donor ligands and their application in dye-sensitized solar cells

Two series of novel ruthenium bipyridyl dyes incorporating sulfur-donor bidentate ligands with general formula \[Ru(R-bpy)2C2N2S2] and \[Ru(R-bpy)2(S2COEt)]\[NO3] (where R =H, CO2Et, CO2H; C2N2S2 = cyanodithioimidocarbonate and S2COEt = ethyl xanthogenate) have been synthesized and characterized spectroscopically, electrochemically and computationally. The acid derivatives in both series (C2N2S2 3 and S2COEt 6) were used as a photosensitizer in a dye-sensitized solar cell (DSSC) and the incident photo-to-current conversion efficiency (IPCE), overall efficiency (_) and kinetics of the dye/TiO2 system were investigated. It was found that 6 gave a higher efficiency cell than 3 despite the latter dye’s more favorable electronic properties, such as greater absorption range, higher molar extinction coefficient and large degree of delocalization of the HOMO. The transient absorption spectroscopy studies revealed that the recombination kinetics of 3 were unexpectedly fast, which was attributed to the terminal CN on the ligand binding to the TiO2, as evidenced by an absorption study of R =H and CO2Et dyes sensitized on TiO2, and hence leading to a lower efficiency DSSC.

Listening to late discovery adoption and donor offspring stories : adoption, ethics and implications for contemporary donor insemination practices

For most of the 20th Century a ‘closed’ system of adoption was practised throughout Australia and other modern Western societies. This ‘closed’ system was characterised by sealed records; amended birth certificates to conceal the adoption, and prohibited contact with all biological family. Despite claims that these measures protected these children from the taint of illegitimacy the central motivations were far more complex, involving a desire to protect couples from the stigma of infertility and to provide a socially acceptable family structure (Triseliotis, Feast, & Kyle, 2005; Marshall & McDonald, 2001). From the 1960s significant evidence began to emerge that many adopted children and adults were experiencing higher incidences of psychological difficulties, characterised by problems with psychological adjustment, building self-esteem and forming a secure personal identity. These difficulties became grouped under the term ‘genealogical bewilderment’. As a result, new policies and practices were introduced to try to place the best interests of the child at the forefront. These changes reflected new understandings of adoption; as not only an individual process but also as a social and relational process that continues throughout life. Secrecy and the withholding of birth information are now prohibited in the overwhelming majority of all domestic adoptions processed in Australia (Marshall & McDonald, 2001). One little known consequence of this ‘closed’ system of adoption was the significant number of children who were never told of their adoptive status. As a consequence, some have discovered or had this information disclosed to them, as adults. The first study that looked at the late discovery of genetic origins experiences was conducted by the Post Adoption Resource Centre in New South Wales in 1999. This report found that the participants in their study expressed feelings of disbelief, confusion, anger, sorrow and loss. Further, the majority of participants continued to struggle with issues arising from this intentional concealment of their genetic origins (Perl & Markham, 1999). A second and more recent study (Passmore, Feeney & Foulstone, 2007) looked at the issue of secrecy in adoptive families as part of a broader study of 144 adult adoptees. This study found that secrecy and/or lies or misinformation on the part of adoptive parents had negative effects on both personal identity and relationships with others. The authors noted that those adoptees who found out about their adoption as adults were ‘especially likely to feel a sense of betrayal’ (p.4). Over recent years, stories of secrecy and late discovery have also started to emerge from sperm donor conceived adults (Spencer, 2007; Turner & Coyle, 2000). Current research evidence shows that although a majority of couples during the donor assisted conception process indicate that they intend to tell the offspring about their origins, as many as two-thirds or more of couples continue to withhold this information from their children (Akker, 2006; Gottlieb, A. McWhinnie, 2001; Salter-Ling, Hunter, & Glover, 2001). Why do they keep this secret? Infertility involves a range of complex factors that are often left unresolved or poorly understood by those choosing insemination by donor as a form of family building (Schaffer, J. A., & Diamond, R., 1993). These factors may only impact after the child is born, when resemblance talk becomes most pronounced. Resemblance talk is an accepted form of public discourse and a social convention that legitimises the child as part of the family and is part of the process of constructing the child’s identity within the family. Couples tend to become focused on resemblance as this is where they feel most vulnerable, and the lack of resemblance to the parenting father may trigger his sense of loss (Becker, Butler, & Nachtigall, 2005).

A Transformational Role : Donor and charity perspectives on major giving in Australia

Tapping into the thoughts of nearly 50 Australians involved with major giving, this study seeks to know more about why and how people give in what might be called ‘momentous’ ways. It tracks both their triumphs and trials. Perhaps most importantly, it gives a public voice to the perceptions, attitudes, concerns and stories of Australians who have chosen to act philanthropically in a sizeable and ongoing way. In counterpoint, the views, experiences and frustrations of seasoned fundraising professionals who work to generate major giving across a range of causes form the other voices in this study. Thus, donors talk about giving, and occasionally raising support from their peers, and fundraisers talk about developing major gifts. This research has been supported by the Perpetual Foundation, the EF and SL Gluyas Trust and the Edward Corbould Charitable Trust under the management of Perpetual Trustee Company Ltd.

Exploring the role of gender and risk perceptions in people’s decisions to register as a bone marrow donor

Increasing the number of bone marrow (BM) donors is important to ensure sufficient diversity on BM registries to meet the needs of patients. This study used an experimental approach to test the hypothesis that providing information about the risks of BM donation to allay unsubstantiated fears would reduce male and female participants’ perceptions of risk for donation and joining the Australian BM Donor Registry (ABMDR). Males’ and females’ intentions to register on the ABMDR, their attitudes, norms, and perceived behavioural control (efficacy) in relation to registering were explored also. Participants were allocated randomly to either a risk (exposed to risk information about BM donation) or no risk(not exposed to risk information) condition. In partial support of hypotheses, exposure to risk information did reduce perceived risk for registering on the ABMDR for males only. Participants in the risk condition also demonstrated lower scores on attitude (males only) and intention compared to participants in the no risk condition. These findings highlight the complex role of risk perceptions and gender differences in understanding people’s decisions to join a BM registry.