Annette Trickett

T cell stimulation and expansion using anti-CD3/CD28 beads

Following appropriate stimulation, T lymphocytes will proliferate extensively in vitro. Traditionally, mitogenic lectins such as phytohemagglutinin (PHA) and concanavalin A (Con A) have been used for polyclonal T cell stimulation. A more physiologically relevant approach uses beads coated with anti-CD3 and anti-CD28 to stimulate T cells in a manner that partially mimics stimulation by antigen-presenting cells. This protocol describes the steps involved in T cell stimulation and their subsequent in vitro expansion using anti-CD3/CD28 beads.

Involvement of cytochrome c release and caspase-3 activation in the oxidative stress-induced apoptosis in human tendon fibroblasts

Our previous studies have demonstrated that oxidative stress and apoptosis are involved in human tendon degeneration. The objectives of our current study were to investigate the effect of oxidative stress on human tendon cell apoptosis, and to explore pathways by which tendon cell apoptosis was induced. In vitro oxidative stress was created by exposure of cultured human rotator cuff tendon cells to H(2)O(2). Apoptotic cells were assessed by Annexin V-FITC staining and necrotic cells by propidium iodide (PI) staining using flow cytometry. Cytochrome c and caspase-3 protein expression were detected by Western blotting. A mini-dialysis unit was employed to increase the protein concentration of the cytosolic fraction. Caspase-3 activity was determined by a colorimetric assay. Tendon cell apoptosis induced by H(2)O(2) was both dose and time dependent. Addition of H(2)O(2) resulted in the release of cytochrome c to the cytosol, and an increase of caspase-3 activity and the expression of caspase-3 subunit. The data suggest that oxidative stress-induced apoptosis in human tendon fibroblasts is mediated via pathway(s) that includes release of cytochrome c from mitochondria to the cytosol and activation of caspase-3.

Expression and regulation of peroxiredoxin 5 in human osteoarthritis.

Reactive oxygen species (ROS) are implicated in the pathogenesis of osteoarthritis (OA). However, little is known about the antioxidant defence system in articular cartilage. We investigated the expression and regulation of peroxiredoxin 5 (PRDX5), a newly discovered thioredoxin peroxidase, in human normal and osteoarthritic cartilage. Our results show that human cartilage constitutively expresses PRDX5. Moreover, the expression is up‐regulated in OA. Inflammatory cytokines tumour necrosis factor α and interleukin 1 β contribute to this up‐regulation by increasing intracellular ROS production. The present study suggests that PRDX5 may play a protective role against oxidative stress in human cartilage.

Ex vivo expansion of functional T lymphocytes from HIV-infected individuals

This study was designed to define the conditions for expansion of functional T lymphocytes from human immunodeficiency virus (HIV)-infected subjects, with the ultimate goal of using these cells for immunotherapy. The most appropriate culture conditions for good T cell proliferation included stimulation with anti-CD3 and anti-CD28 coated microspheres, and propagation in Aim V serum-free media with 20 U/ml interleukin-2 (IL-2), supplemented with decreasing concentrations of serum for the initial 8 days. Under these conditions, a 14-day culture period yielded approximately a 10,000-fold expansion of T lymphocytes from HIV-infected donors. The cultured cells comprised approximately 15% CD4+ cells and 70% CD8+ cells. These cells retained functional capacity as assessed by cytotoxicity towards HIV proteins, and production of IL-2 and interferon-gamma (IFN-gamma). Viral replication within the culture system was controlled, but not eliminated, without the requirement for antiviral agents. These culture conditions were demonstrated to be suitable for larger scale expansion of cells in hollow fibre bioreactors. This methodology provides a suitable means of producing large quantities of functional T cells for use in autologous immunotherapy protocols.

Regulation of cellular therapy in Australia

Summary Use of cellular products for therapeutic purposes has predominantly been unregulated in Australia until recently. Transplant of haemopoietic progenitor cells (HPC) for bone marrow regeneration is now a routine treatment for many disorders with an established mechanism of facility accreditation. However, other cellular therapies do not have any form of accreditation, are not well evaluated and may be associated with significant risks. On 31 May 2011 the Therapeutic Goods Administration (TGA) implemented a long heralded regulatory biologicals framework for cell and tissue based therapies. The framework currently excludes human HPC, organs for direct transplantation and reproductive materials which are already covered by various forms of existing peer review and accreditation. This new framework is a practical approach for applying regulation based on the risk of the product to the recipient with four classes of product. Class 1 is reserved for the least regulated products and currently does not contain any proposed products. Class 2 will be for minimally manipulated products which will only require manufacturing compliance and evaluation against product and other mandatory standards before entry onto the Australian Register of Therapeutic Goods (ARTG). Class 3 and 4 products will be more than minimally manipulated and these cells and tissues may be used in a non-homologous manner. Class 3 and 4 products will represent a spectrum of risk where Class 4 therapies will represent the highest potential risk to the recipient, with the same requirements for Class 2 approvals but with additional requirements for comprehensive evaluation of a dossier for quality, safety and efficacy of the product. The extent of this quality, safety and efficacy data will depend upon the nature of the product and its associated risks, but will be more comprehensive for Class 4 as opposed to Class 3 products. The only truly contentious feature of this framework is the extremely high cost for dossier evaluation and the puzzling absence of an orphan drug scheme for biologicals.

A preliminary study to determine the effect of an infusion of cryopreserved autologous lymphocytes on immunocompetence and viral load in HIV-infected patients

Therapeutic measures aimed at boosting the immunity of HIV-infected patients are a critical component of strategies for effective therapy of HIV and AIDS. To improve immunocompetence in patients with progressive disease, autologous lymphocytes that were collected and cryopreserved earlier in the course of HIV-infection were reinfused. None of the 12 patients receiving cell infusions experienced any adverse effects. Improvements in immunologic parameters (CD4+ counts, CD8+ counts, or both; HIV-specific cytotoxic T-lymphocyte (CTL) activity; or viral load) were seen in seven patients. Restoration of the CD4+ count to the level recorded at the time of cell harvest was achieved in two patients with less advanced disease. Plasma HIV RNA was reduced by >0.5 logs in two of the four patients tested. These preliminary results suggest that cellular immunotherapy using cryopreserved autologous lymphocytes has the potential to improve some measures of immunity in patients with HIV/AIDS and warrants further investigation.

Optimization of microbial screening for cord blood.

Collection and processing of cord blood (CB) is associated with significant risk of contamination; hence standards mandate microbial screening of the final product. The sensitivity of current methods to evaluate the microbial content of CB is unknown, given the small volume tested and reduced sensitivity of pediatric bottles. Hence, this study was undertaken to evaluate an optimal microbial screening method.

Effects of cryopreservation on microbial‐contaminated cord blood

Cord blood units (CBUs) are associated with significant risk of exposure to microbial contamination during collection and processing; however, the survival of bacteria within a CBU is poorly understood. This study aimed to determine whether contaminating organisms in CBU survive the cryopreservation, frozen storage, and subsequent thawing conditions before infusion.

Flexible low-intensity combination chemotherapy for elderly patients with acute myeloid leukemia.

Twenty-five patients aged 57 to 88 years (median, 70 years) with acute myeloid leukemia were treated with a flexible low-intensity treatment regimen comprising mitozantrone (mitoxantrone) 6 mg/m2 administered by intravenous infusion ×3 days, cytarabine 10 mg/m2 subcutaneously every 12 hours ×7 to 14 days, and etoposide 100 mg orally ×7 to 14 days. Seventeen of these patients had a preexisting myelodysplastic syndrome. The clinical response was correlated to the results of cytogenetic studies (23 patients) and of viability studies of leukemic blasts (7 patients). Eleven of the 25 patients achieved complete remission (CR), 8 achieved partial remission (PR), and 4 showed no response. There was 1 toxic death, and 1 patient died soon (1 week) after presentation. Treatment was well tolerated. Although myelotoxicity occurred regularly, the recovery time was ≤3 weeks for most of the responding patients. Duration of survival for patients who had CR has ranged from 4+ to 43+ months and for patients who had PR, 3 to 16 months. Irrespective of the remission status (CR or PR), responding patients with favorable (n = 1) or intermediate (n = 10) cytogenetic findings had a significantly better survival time (median, 14 months) than did those with unfavorable (n = 7) cytogenetic findings (median, 5 months). In vitro studies showed a progressive reduction in the number of circulating blasts. The number of viable blasts 3 days after initiation of therapy appeared to give an early indication of clinical response. Treatment with a flexible low-intensity protocol seems to achieve results comparable with those reported for intensive antileukemia therapy and has much less toxicity.

Haematopoietic stem cell transplantation for primary immunodeficiency syndromes: A 5-year single-centre experience

Haematopoietic stem cell transplantation (HSCT) is a central therapy in the treatment of primary immunodeficiency diseases (PIDs). Over the past 5 years, outcomes have been greatly improved due to earlier diagnosis, improved donor availability, advancements in graft manipulation and the use of less toxic preparative regimens. We present a 5‐year audit of HSCT for PID at a single Australian tertiary hospital.