Closed system isolation and scalable expansion of human placental mesenchymal stem cells

Mesenchymal stem cells (MSC) are emerging as a leading cellular therapy for a number of diseases. However, for such treatments to become available as a routine therapeutic option, efficient and cost-effective means for industrial manufacture of MSC are required. At present, clinical grade MSC are manufactured through a process of manual cell culture in specialized cGMP facilities. This process is open, extremely labor intensive, costly, and impractical for anything more than a small number of patients. While it has been shown that MSC can be cultivated in stirred bioreactor systems using microcarriers, providing a route to process scale-up, the degree of numerical expansion achieved has generally been limited. Furthermore, little attention has been given to the issue of primary cell isolation from complex tissues such as placenta. In this article we describe the initial development of a closed process for bulk isolation of MSC from human placenta, and subsequent cultivation on microcarriers in scalable single-use bioreactor systems. Based on our initial data, we estimate that a single placenta may be sufficient to produce over 7,000 doses of therapeutic MSC using a large-scale process.

In vitro primary cell culture as a physiologically relevant method for preclinical testing of human oncolytic adenovirus

Ad[I/PPT-E1A] is an oncolytic adenovirus that specifically kills prostate cells via restricted replication by a prostate-specific regulatory element. Off-target replication of oncolytic adenoviruses would have serious clinical consequences. As a proposed ex vivo test, we describe the assessment of the specificity of Ad[I/PPT-E1A] viral cytotoxicity and replication in human nonprostate primary cells. Four primary nonprostate cell types were selected to mimic the effects of potential in vivo exposure to Ad[I/PPT-E1A] virus: bronchial epithelial cells, urothelial cells, vascular endothelial cells, and hepatocytes. Primary cells were analyzed for Ad[I/PPT-E1A] viral cytotoxicity in MTS assays, and viral replication was determined by hexon titer immunostaining assays to quantify viral hexon protein. The results revealed that at an extreme multiplicity of infection of 500, unlikely to be achieved in vivo, Ad[I/PPT-E1A] virus showed no significant cytotoxic effects in the nonprostate primary cell types apart from the hepatocytes. Transmission electron microscopy studies revealed high levels of Ad[I/PPT-E1A] sequestered in the cytoplasm of these cells. Adenoviral green fluorescent protein reporter studies showed no evidence for nuclear localization, suggesting that the cytotoxic effects of Ad[I/PPT-E1A] in human primary hepatocytes are related to viral sequestration. Also, hepatocytes had increased amounts of coxsackie adenovirus receptor surface protein. Active viral replication was only observed in the permissive primary prostate cells and LNCaP prostate cell line, and was not evident in any of the other nonprostate cells types tested, confirming the specificity of Ad[I/PPT-E1A]. Thus, using a relevant panel of primary human cells provides a convenient and alternative preclinical assay for examining the specificity of conditionally replicating oncolytic adenoviruses in vivo.

Gene transfer vectors targeted to human prostate cancer : do we need better preclinical testing systems?

Destruction of cancer cells by genetically modified viral and nonviral vectors has been the aim of many research programs. The ability to target cytotoxic gene therapies to the cells of interest is an essential prerequisite, and the treatment has always had the potential to provide better and more long-lasting therapy than existing chemotherapies. However, the potency of these infectious agents requires effective testing systems, in which hypotheses can be explored both in vitro and in vivo before the establishment of clinical trials in humans. The real prospect of off-target effects should be eliminated in the preclinical stage, if current prejudices against such therapies are to be overcome. In this review we have set out, using adenoviral vectors as a commonly used example, to discuss some of the key parameters required to develop more effective testing, and to critically assess the current cellular models for the development and testing of prostate cancer biotherapy. Only by developing models that more closely mirror human tissues will we be able to translate literature publications into clinical trials and hence into acceptable alternative treatments for the most commonly diagnosed cancer in humans.

Students’ beliefs about willingness to access complementary and alternative therapies (CAT) training for future integration into psychology practice.

It is suggested that all psychologists gain basic training in the types of complementary and alternative therapies (CAT) their clients may be using. As psychology students are the next cohort of health professionals who will inform future initiatives in the field, it is important to first understand the factors which influence their decisions about CAT integration. Drawing on the Theory of Planned Behavior, we investigated the beliefs that differentiate between psychology students who are high or low on willingness to access training in CAT for future practice use. Psychology students (N = 106) completed a questionnaire assessing the likelihood of both positive and negative consequences of accessing training and utilizing CAT within a psychological practice, important others approval, and barriers preventing them from this integration behavior. Those students high compared to low on willingness more likely to endorse positive outcomes (e.g., offering a more holistic approach to therapy) of accessing CAT training for future practice use and to believe that important others (e.g., clients) would support this behavior. We identified important beliefs of student psychologists related to decisions about undertaking CAT training for future professional use and can inform educators and policy-makers about CAT training and integration in psychology practice.

Hormone-dependent bacterial growth persistence and biofilm formation - A pilot study investigating human follicular fluid collected during IVF cycles

Human follicular fluid, considered sterile, is aspirated as part of an in vitro fertilization (IVF) cycle. However, it is easily contaminated by the trans-vaginal collection route and little information exists in its potential to support the growth of microorganisms. The objectives of this study were to determine whether human follicular fluid can support bacterial growth over time, whether the steroid hormones estradiol and progesterone (present at high levels within follicular fluid) contribute to the in vitro growth of bacterial species, and whether species isolated from follicular fluid form biofilms. We found that bacteria in follicular fluid could persist for at least 28 weeks in vitro and that the steroid hormones stimulated the growth of some bacterial species, specifically Lactobacillus spp., Bifidobacterium spp. Streptococcus spp. and E. coli. Several species, Lactobacillus spp., Propionibacterium spp., and Streptococcus spp., formed biofilms when incubated in native follicular fluids in vitro (18/24, 75%). We conclude that bacteria aspirated along with follicular fluid during IVF cycles demonstrate a persistent pattern of growth. This discovery is important since it can offer a new avenue for investigation in infertile couples.

INT6/EIF3E interacts with ATM and is required for proper execution of the DNA damage response in human cells

Altered expression of the INT6 gene, encoding the e subunit of the translational initiation factor eIF3, occurs in human breast cancers, but how INT6 relates to carcinogenesis remains unestablished. Here, we show that INT6 is involved in the DNA damage response. INT6 was required for cell survival following γ-irradiation and G(2)-M checkpoint control. RNA interference-mediated silencing of INT6 reduced phosphorylation of the checkpoint kinases CHK1 and CHK2 after DNA damage. In addition, INT6 silencing prevented sustained accumulation of ataxia telangiectasia mutated (ATM) at DNA damage sites in cells treated with γ-radiation or the radiomimetic drug neocarzinostatin. Mechanistically, this result could be explained by interaction of INT6 with ATM, which together with INT6 was recruited to the sites of DNA damage. Finally, INT6 silencing also reduced ubiquitylation events that promote retention of repair proteins at DNA lesions. Accordingly, accumulation of the repair factor BRCA1 was defective in the absence of INT6. Our findings reveal unexpected and striking connections of INT6 with ATM and BRCA1 and suggest that the protective action of INT6 in the onset of breast cancers relies on its involvement in the DNA damage response.

Motorcycle Safety Research Project Interim Summary Report 3 : Training and Licensing Interventions for Risk Taking and Hazard Perception for Motorcyclists

Motorcycle trauma is a serious road safety issue in Queensland and throughout Australia. In 2009, Queensland Transport (later Transport and Main Roads or TMR) appointed CARRS-Q to provide a three-year program of Road Safety Research Services for Motorcycle Rider Safety. Funding for this research originated from the Motor Accident Insurance Commission. This program of research was undertaken to produce knowledge to assist TMR to improve motorcycle safety by further strengthening the licensing and training system to make learner riders safer by developing a pre-learner package (Deliverable 1), and by evaluating the QRide CAP program to ensure that it is maximally effective and contributes to the best possible training for new riders (Deliverable 2). The focus of this report is Deliverable 3 of the overall program of research. It identifies potential new licensing components that will reduce the incidence of risky riding and improve higher-order cognitive skills in new riders.

Suspected child abuse and neglect reporting behaviour

Executuve Summary Background and Aims Child abuse and neglect is a tragedy within our community, with over 10,000 substantiated reports of abuse and neglect in Queensland in the past year. The considerable consequences of child abuse and neglect are far-reaching, substantial and can be fatal. The reporting of suspicions of child abuse or neglect is often the first step in preventing further abuse or neglect. In the State of Queensland, medical practitioners are mandated by law to report their suspicions of child abuse and neglect. However, despite this mandate many still do not report their suspicions. A 1998 study indicated that 43% of medical practitioners had, at some time, made a conscious decision to not report suspected abuse or neglect (Van Haeringen, Dadds & Armstrong, 1998). The aim of this study was to gain a better understanding of beliefs about reporting suspected child abuse and neglect and the barriers to reporting suspected abuse and neglect by medical practitioners and parents and students. The findings have the potential to inform the training and education of members of the community who have a shared responsibility to protect the wellbeing of its most vulnerable members. Method In one of the largest studies of reporting behaviour in relation to suspected child abuse and neglect in Australia, we examined and compared medical practitioners’ responses with members of the community, namely parents and students. We surveyed 91 medical practitioners and 214 members of the community (102 parents and 112 students) regarding their beliefs and reporting behaviour related to suspected child abuse and neglect. We also examined reasons for not reporting suspected abuse or neglect, as well as awareness of responsibilities and the appropriate reporting procedures. To obtain such information, participants anonymously completed a comprehensive questionnaire using items from previous studies of reporting attitudes and behaviour. Executive Summary Abused Child Trust Report August 2003 5 Findings Key findings include: • The majority of medical practitioners (97%) were aware of their duty to report suspected abuse and neglect and believed they had a professional and ethical duty to do so. • A majority of parents (82%) and students (68%) also believed that they had a professional and ethical duty to report suspected abuse and neglect. • In accord with their statutory duty to report suspected abuse and neglect, 69% of medical practitioners had made a report at some point. • Sixteen percent of parents and 9% of students surveyed indicated that they had reported their suspicions of neglect and abuse. • The most endorsed belief associated with not reporting suspected child abuse and neglect was that, ‘unpleasant events would follow reporting’. • Over a quarter of medical practitioners (26%) admitted to making a decision not to report their suspicions of child abuse or neglect on at least one occasion. • Compared with previous research, there has been a decline in the number of medical practitioners who decided not to report suspected abuse or neglect from 43% (Van Haeringen et al., 1998) to 26% in the current study. • Fourteen percent of parents and 15% of students surveyed had also chosen not to report a case of suspected abuse or neglect. • Attitudes that most strongly influenced the decision to report or not report suspected abuse or neglect differed between groups (medical practitioners, parents, or students). A belief that, ‘the abuse was a single incident’ was the best predictor of non-reporting by medical practitioners, while having ‘no time to follow-up the report’ or failing to be ‘convinced of evidence of abuse’ best predicted failure to report abuse by students. A range of beliefs predicted non-reporting by parents, including the beliefs that reporting suspected abuse was ‘not their responsibility’ and ‘knowing the child had retracted their statement’. Conclusions Of major concern is that approximately 25% of medical practitioners with a mandated responsibility to report, as well as some members of the general public, revealed that they have suspected child neglect or abuse but have made the decision not to report their suspicions. Parents and students perceived the general community as having responsibility for reporting suspicions of abuse or neglect. Despite this perception, they felt that lodging a report may be overly demanding in terms of time and they had the confidence in their ability to identify child abuse and neglect. An explanation for medical practitioners deciding not to report may be based upon their optimistic belief that suspected abuse or neglect was a single incident. Our findings may best be understood from the ‘inflation of optimism’ hypothesis put forward by the Nobel Laureate, Daniel Kahneman. He suggests that in spite of rational evidence, human beings tend to make judgements based on an optimistic view rather than engaging in a rational decision-making process. In this case, despite past behaviour of abuse or neglect being the best predictor of future behaviour, medical practitioners have taken an optimistic view, choosing to believe that their suspicion of child abuse or neglect represents a single incident. The clear implication of findings in the current research is the need for the members of the general community and medical practitioners to be better appraised of the consequences of their decision-making in relation to suspicionsof child abuse and neglect. Finally findings from parents and students relating to their reporting behaviour suggest that members of the larger community represent an untapped resourcewho might, with appropriate awareness, play a more significant role in theidentification and reporting of suspected child abuse and neglect.

Nagelschmidtite bioceramics with osteostimulation properties : material chemistry activating osteogenic genes and WNT signalling pathway of human bone marrow stromal cells

Bioactive materials with osteostimulation properties are of great importance to promote osteogenic differentiation of human bone marrow stromal cells (hBMSCs) for potential bone regeneration. We have recently synthesized nagelschmidtite (NAGEL, Ca7Si2P2O16) ceramic powders which showed excellent apatite-mineralization ability. The aim of this study was to investigate the interaction of hBMSCs with NAGEL bioceramic bulks and their ionic extracts, and to explore the osteostimulation properties of NAGEL bioceramics and the possible molecular mechanism. The cell attachment, proliferation, bone-related gene expression (ALP, OPN and OCN) and WNT signalling pathways (WNT3a, FZD6, AXIN2 and CTNNB) of hBMSCs cultured on NAGEL bioceramic disks were systematically studied. We further investigated the biological effects of ionic products from NAGEL powders on cell proliferation and osteogenic differentiation of hBMSCs by culturing cells with NAGEL extracts. Furthermore, the effect of NAGEL bioceramics on the osteogenic differentiation in hBMSCs was also investigated with the addition of cardamonin, a WNT inhibitor. The results showed that NAGEL bioceramic disks supported the attachment and proliferation of hBMSCs, and significantly enhanced the bone-related gene expression and WNT signalling pathway of hBMSCs, compared to conventional beta-tricalcium phosphate (β-TCP) bioceramic disks and blank controls. The ionic products from NAGEL powders also significantly promoted the proliferation, bone and WNT-related gene expression of hBMSCs. It was also identified that NAGEL bioceramics could bypass the action of the WNT inhibitor (10 μM) to stimulate the selected osteogenic genes in hBMSCs. Our results suggest that NAGEL bioceramics possess excellent in vitro osteostimulation properties. The possible mechanism for the osteostimulation may be directly related to the released Si, Ca and P-containing ionic products from NAGEL bioceramics which activate bone-related gene expression and WNT signalling pathway of hBMSCs. The present study suggests that NAGEL bioceramics are a potential bone regeneration material with significant osteostimulation capacity.

Training opportunities in public art

Craft practitioners and designers have been involved in the production of civic art commissions for many centuries, either as recognised art workers or anonymous artisans. However, it has only been in the last couple of decades that Australia has formally acknowledged the role of public art through the development of government policy and professional accredited training

Effect of bone morphogenetic protein-4 (BMP-4) on the expression of Sox2, Oct-4 and c-Myc in human periodontal ligament cells during long-term culture

Recent studies demonstrated endogenous expression level of Sox2, Oct-4 and c-Myc is correlated with the pluripotency and successful induction of induced pluripotent stem cells (iPSCs). Periondontal ligament cells (PDLCs)have multi-lineage diferentiation capability and ability to maintain undifferentiated stage, which makes PDLCs a suitable cell source for tissue repair and regeneration. To elucidate the effect of in vitro culture condition on the stemness potential of PDLCs, we explored the cell growth, proliferation, cell cycle, and the expression of Sox2, Oct-4 and c-Myc in PDLCs from passage 1 to 7 with or without the addition of recombinant human BMP4(rhBMP4). Our results revealed that BMP-4 promoted cell growth and proliferation, arrested PDLCs in S phase of cell cycle and upregulated PI value. It was revealed that without the addition of rhBMP4, the expression of Sox2, Oct-4 and c-Myc in PDLCs only maintained nucleus location until passage 3, then lost nucleus location subsequently. The mRNA expression in PDLCs further confirmed that the level of Sox2 and Oct-4 peaked at passage 3, then decreased afterwards, whereas c-Myc maintained consistently upregulation along passages. after the treatment with rhBMP4, the expression of Sox2, Oct-4 and c-Myc in PDLCs maintained nucleus location even at passage 7 and the mRNA expression of Sox2 and Oct-4 significantly upregulated at passage 5 and 7. These results demonstrated that addition of rhBMP-4 in the culture media could improve the current culture condition for PDLCs to maintain in an undifferentiated stage.

The human dimension of nosocomial wound infection : a study in liminality

Nosocomial wound infection is a disease that has to date been primarily understood through the language of science and biomedicine. This paper reports on findings from a sociological, interpretive study that focused on the experiential dimension of this phenomenon. The illness experience of a nosocomial wound infection is examined within a cultural milieu that values the smooth, untroubled body and alternatively ascribes cultural meaning to a body that has a definable illness. Within this context the person with a chronic wound from nosocomial infection defies normative categorisation and is thus situated outside the patterning of society. The human dimension of nosocomial wound infection includes the private, existential and embodied aspects of living with a chronic, infected wound. This report indicates that the experiential dimension is characterised by an embodied state of liminality. People with this illness live an indeterminate existence that is in-between health and illness, cure and disease. As such they have no recognised place in the medical or social world.

Rail incident investigator training: Is there support for a standardised Australasian training framework?

Background: Historically rail organisations have been operating in silos and devising their own training agendas. However with the harmonisation of the Australian workplace health and safety legislation and the appointment of a national rail safety regulator in 2013, rail incident investigator experts are exploring the possibility of developing a unified approach to investigator training. Objectives: The Australian CRC for Rail Innovation commissioned a training needs analysis to identify if common training needs existed between organisations and to assess support for the development of a national competency framework for rail incident investigations. Method: Fifty-two industry experts were consulted to explore the possibility of the development of a standardised training framework. These experts were sourced from within 19 Australasian organisations, comprising Rail Operators and Regulators in Queensland, New South Wales, Victoria, Western Australia, South Australia and New Zealand. Results: Although some competency requirements appear to be organisation specific, the vast majority of reported training requirements were generic across the Australasian rail operators and regulators. Industry experts consistently reported strong support for the development of a national training framework. Significance: The identification of both generic training requirements across organisations and strong support for standardised training indicates that the rail industry is receptive to the development of a structured training framework. The development of an Australasian learning framework could: increase efficiency in course development and reduce costs; establish recognised career pathways; and facilitate consistency with regards to investigator training.

Employee perceptions regarding the effectiveness of occupational road safety initiatives

A range of risk management initiatives have been introduced in organisations in attempt to reduce occupational road incidents. However a discrepancy exists between the initiatives that are frequently implemented in organisations and the initiatives that have demonstrated scientific merit in improving occupational road safety. Given that employees’ beliefs may facilitate or act as a barrier to implementing initiatives, it is important to understand whether initiatives with scientific merit are perceived to be effective by employees. To explore employee perceptions pertaining to occupational road safety initiatives, a questionnaire was administered to 679 employees sourced from four Australian organisations. Participants ranged in age from 18 years to 65 years (M = 42, SD = 11). Participants rated 35 initiatives based on how effective they thought they would be in improving road safety in their organisation. The initiatives perceived by employees to be most effective in managing occupational road risks comprised: making vehicle safety features standard e.g. passenger airbags; practical driver skills training; and investigation of serious vehicle incidents. The initiatives perceived to be least effective in managing occupational road risks comprised: signing a promise card commitment to drive safely; advertising the organisation’s phone number on vehicles for complaints and compliments; and consideration of driving competency in staff selection process. Employee perceptions were analysed at a factor level and at an initiative level. The mean scores for the three extracted factors revealed that employees believed occupational road risks could best be managed by the employer implementing engineering and human resource methods to enhance road safety. Initiatives relating to employer management of identified risk factors were perceived to be more effective than feedback or motivational methods that required employees to accept responsibility for their driving safety. Practitioners can use the findings from this study to make informed decisions about how they select, manage and market occupational safety initiatives.