Genome-wide transcription factor-binding maps reveal cell-specific changes in the regulatory architecture of human HSPCs.

Hematopoietic stem and progenitor cells (HSPCs) rely on a complex interplay among transcription factors (TFs) to regulate differentiation into mature blood cells. A heptad of TFs (FLI1, ERG, GATA2, RUNX1, TAL1, LYL1, LMO2) bind regulatory elements in bulk CD34+ HSPCs. However, whether specific heptad-TF combinations have distinct roles in regulating hematopoietic differentiation remains unknown. We mapped genome-wide chromatin contacts (HiC, H3K27ac, HiChIP), chromatin modifications (H3K4me3, H3K27ac, H3K27me3) and 10 TF binding profiles (heptad, PU.1, CTCF, STAG2) in HSPC subsets (stem/multipotent progenitors plus common myeloid, granulocyte macrophage, and megakaryocyte erythrocyte progenitors) and found TF occupancy and enhancer-promoter interactions varied significantly across cell types and were associated with cell-type-specific gene expression. Distinct regulatory elements were enriched with specific heptad-TF combinations, including stem-cell-specific elements with ERG, and myeloid- and erythroid-specific elements with combinations of FLI1, RUNX1, GATA2, TAL1, LYL1, and LMO2. Furthermore, heptad-occupied regions in HSPCs were subsequently bound by lineage-defining TFs, including PU.1 and GATA1, suggesting that heptad factors may prime regulatory elements for use in mature cell types. We also found that enhancers with cell-type-specific heptad occupancy shared a common grammar with respect to TF binding motifs, suggesting that combinatorial binding of TF complexes was at least partially regulated by features encoded in DNA sequence motifs. Taken together, this study comprehensively characterizes the gene regulatory landscape in rare subpopulations of human HSPCs. The accompanying data sets should serve as a valuable resource for understanding adult hematopoiesis and a framework for analyzing aberrant regulatory networks in leukemic cells.

A comprehensive human leukocyte antigen analysis of 36 782 cord blood units stored in the Australian Public Cord Blood Banking Network.

BACKGROUND AND AIMS: The network of public cord blood banks (CBBs) in Australia, known as AusCord, comprises CBBs located in Brisbane, Sydney and Melbourne. A novel comprehensive analysis has been performed to determine whether the cryopreserved, searchable cord blood unit (CBU) inventory of approximately 36 000 units share similar tissue types or haplotypes. METHODS: Human leukocyte antigen (HLA) data was analysed using Microsoft Excel following standardisation of typing data. RESULTS: The analysis has found that the majority of stored, searched and released CBU exhibit a tissue type that is unique within and between the CBBs. Therefore, each collection performed by the CBBs is likely to comprise a tissue type that is not already stored among the total AusCord inventory. HLA alleles (HLA-A*34, HLA-B*56, HLA-DRB1*08:03), which are uncommon in European populations, were associated with Pacific Islander and/or Indigenous Australian populations and confirmed to be more frequent among donors who, when screened, self-identified as these ethnicities. CONCLUSIONS: These data indicate that (i) continued addition of CBU to existing inventories is likely to further increase the HLA diversity and (ii) screening donors for ethnicity or strategically locating collection sites where ethnic minorities reside can successfully result in collection of rare HLA associated with ethnic minority groups for whom finding donors might otherwise be more difficult.

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

AIM: 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. METHODS: Retrospective case note review identified diagnosis, pre-transplant medical morbidity, transplant protocol, engraftment, adverse events, post-transplant immune reconstitution and general health. RESULTS: A total of 22 patients with PID underwent 24 HSCTs at our institution between 2012 and 2016. The most common indications were severe combined immunodeficiency, chronic granulomatous disease and familial haemophagocytic lymphohistiocytosis, with a genetic diagnosis in all but two patients. Reduced intensity or reduced toxicity conditioning was used in 91% of cases, and 75% of the donors were unrelated. Transplant-related mortality at day +100 was 9.5%, and cumulative overall survival was 86%. There were three mortalities, all secondary to viral infection, one of which occurred in the context of graft failure. Two patients remained on immune support, with the remainder achieving adequate immune reconstitution. CONCLUSIONS: The outcomes for HSCT for PIDs performed at Sydney Children's Hospital were in line with the world's best practice. HSCT should be considered a potential therapeutic option for all Australian PID patients with a valid disease indication.

Effects of cryopreservation on microbial-contaminated cord blood.

BACKGROUND: 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. STUDY DESIGN AND METHODS: A total of 134 CBUs rejected from banking due to known contamination were thawed and rescreened using blood culture bottles (BacT/ALERT, bioMérieux). An additional 61 fresh CBUs were deliberately spiked with a range of microbial organisms and evaluated both before freeze and after thaw. RESULTS: Microbial contaminants were detected after thaw in 63% of stored contaminated CBUs and 85% of spiked CBUs. Postthaw organism detection in spiked cord blood (CB) was higher in adult culture bottles (80%) than pediatric culture bottles (61%). Twenty percent of spiked organisms, particularly Bacillus subtilis, Escherichia coli, Clostridium sporogenes, and Propionibacterium acnes, were not detected in prefreeze samples but were detectable after thaw. CONCLUSIONS: This study demonstrates that the majority of contaminating organisms isolated in a prefreeze sample of CB have the ability to survive cryopreservation, frozen storage, and thawing. Further, CBUs reported as microbial free may contain microbial contamination, which could result in transplantation of contaminated CB and be potentially deleterious to a patient.

Optimization of microbial screening for cord blood.

BACKGROUND: 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. STUDY DESIGN AND METHODS: CB was collected using a closed system then spiked with organisms at 1 or 10 colony-forming units (CFUs)/mL. Samples were screened using culture bottles (BacT/ALERT, bioMérieux; and BACTEC, Becton Dickinson). Several methods were evaluated with different combinations of inoculated bottles (adult vs. pediatric), sample types (plasma discard, red blood cell [RBC] discard, or final product), and sample volumes. RESULTS: Of 94 cord blood units (CBUs) spiked with organisms before screening, 81% tested positive for contamination overall. Screening of CB in pediatric bottles resulted in equivalent detection rates on the BacT/ALERT and BACTEC systems (33% at 1 CFU/mL and 73% at 10 CFUs/mL, respectively). However, the pediatric bottle screen only detected 15% of obligate anaerobes. A combined fraction method showed superior detection (71%) compared to the plasma fraction (27%) and resulted in optimal anaerobic detection. CONCLUSIONS: This study demonstrates that the optimal microbial screening method for CB includes testing a combination of discard fractions (plasma and RBCs) in addition to final product using an automated culture system. Inoculating a small sample of final product in a pediatric bottle is suboptimal for microbial detection and may lead to distribution of contaminated CB for transplantation.

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.

Overexpression of antioxidant enzyme peroxiredoxin 5 protects human tendon cells against apoptosis and loss of cellular function during oxidative stress.

Oxidative stress and apoptosis are implicated in tendon degeneration. Peroxiredoxin 5 (PRDX5) is a novel thioredoxin peroxidase recently identified in mammals, participating directly in eliminating hydrogen peroxide (H(2)O(2)) and neutralizing other reactive oxygen species (ROS). We have previously reported that PRDX5 is upregulated in degenerative human tendon. However, the effects of this upregulation on human tendon cell function remain unknown, in particular, with regards to oxidative stress conditions. Here we report that exposure of human tendon cells to 50 microM H(2)O(2) for 24 h (in vitro oxidative stress) caused a significant increase in the percentage of apoptotic cells (P<0.05) as assessed by flow cytometric analysis of Annexin V binding, accompanied by increased PRXD5 mRNA and protein expression. Overexpression of PRDX5 in human tendon cells via transfection inhibited H(2)O(2)-induced tendon cell apoptosis by 46% (P<0.05), and prevented the decrease in tendon cell collagen synthesis which occurs under H(2)O(2) challenge, although the decrease in collagen synthesis was small. Results from our study indicate that the antioxidant enzyme PRDX5 plays a protective role in human tendon cells against oxidative stress by reducing apoptosis and maintaining collagen synthesis.

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 alpha and interleukin 1 beta 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.

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.

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.

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 x3 days, cytarabine 10 mg/m2 subcutaneously every 12 hours x7 to 14 days, and etoposide 100 mg orally x7 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 < or = 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.

Regulation of cellular therapy in Australia.

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.

Platelet activation in the hypertensive disorders of pregnancy.

OBJECTIVE: The purpose of this study was to determine whether platelet activation occurs only in preeclampsia or also in normal pregnancy. STUDY DESIGN: Thirty women with preeclampsia, 30 women with gestational hypertension, 20 women with essential hypertension, 30 pregnant women with normotension, and 30 nonpregnant women were recruited at St George Hospital, Sydney, Australia. Platelet activation was determined by flow cytometry on whole blood samples. RESULTS: Platelet activation was similar in all groups, except the group with preeclampsia. Compared with normal pregnant women, women with preeclampsia had significantly greater CD62 expression (1.35% vs 0.61%; P =.002), CD63 expression (1.73% vs 0.95%; P <.0001) and annexin V binding (1.03% vs 0.66%;P =.03) and significantly fewer circulating platelet microparticles (33 vs 49 x10(9)/L; P =.001). This was unrelated to other parameters that included platelet counts. Women with gestational hypertension in whom preeclampsia developed did not have enhanced platelet activation profiles. CONCLUSION: Platelet activation is increased in preeclampsia but not in other hypertensive disorders or in normal pregnancy. This may be part of the pathophysiologic factors of preeclampsia complications but is not predictable by the platelet count and is not apparent in all women with preeclampsia.