Julia Marley

Cytokine gene therapy or infusion as treatment for solid human cancer

In the induction of tissue-directed immune responses, cytokines tend to be released within the affected tissues. We used two strategies to expose tumor tissues to continuous high levels of cytokines: First, a vaccinia interleukin (IL)2 recombinant was injected directly intratumorally 3-weekly at 10(7) pfus/dose in six patients with the solid tumor malignant mesothelioma (MM). No virus excretion was detectable. At each cycle vaccinia-IL-2 mRNA (SQ [semi-quantitative] reverse transcription polymerase chain reaction) was maximal 24-72 h following injection reduced at 8 days and faded by 21 days. No tumor regression occurred. Second, based on the success of granulocyte macrophage colony-stimulating factor (GM-CSF) in gene transfer experiments, we conducted a study using continuous intratumoral GM-CSF infusion in eight patients with MM using a portable pump at doses of 10 micro/cg/24 h over 8 weeks. Systemic neutrophil agglutination and local catheter-related difficulties occurred. Two patients demonstrated tumor necrosis, one of whom had a marked progressive mononuclear cell infiltration of the tumor associated with a partial response (>50% reduction in tumor area). Murine studies using our MM model in CBA and BALB/C mice have demonstrated that B7-1 and allo-class I transfections induce strong tumor-specific cytotoxic T lymphocyte responses: GM-CSF, IL-12, and IL-2 induced mixed nonspecific plus specific responses, whereas B7-2 and class II transfections were not effective. We conclude that increased intratumoral cytokine concentrations can be generated using both gene transfer and cytokine infusion approaches; however, both have their limitations and, at this stage, have not produced dramatic antitumor effects in humans.

Notch1 co-localizes with CD4 on activated T cells and Notch signaling is required for IL-10 production

The effector function of activated CD4+ T cells and secretion of cytokines are important in the establishment of productive immune responses and tolerance. We identified expression by CD4+ T cells of Notch receptors and ligands and enhanced Notch signaling upon activation. Notch1 expression was up regulated and co‐localized with CD4 upon T cell stimulation. Disruption of Notch signaling did not affect proliferation, but attenuated cytokine secretion following CD3 ligation in the absence of anti‐CD28 antibody. Notch signaling was absolutely necessary for transcription of IL‐10 by stimulated CD4+ T cells. CD4+ T cells transfected with constitutively active Notch1 failed to proliferate, but exhibited enhanced cytokine secretion upon stimulation. Our data indicates that Notch receptor signaling can influence both proliferative and cytokine responses of CD4+ T cells. In addition, the finding that Notch signaling is required for production of IL‐10 may allude to a role in immune regulation.

Transfection of the gene for B7-1 but not B7-2 can induce immunity to murine malignant mesothelioma

Transfection of the genes encoding the co‐stimulatory molecules B7‐1 and B7‐2 has enhanced the development of immunity to a variety of experimental tumors, although most of these were inherently immunogenic. We have determined the effect of expression of these genes on the induction of immunity to 2 non‐immunogenic murine malignant mesothelioma (MM) cell lines (AC29 and AB1). We had previously shown that B7‐1 transfection into AC29 delayed but did not prevent tumor development by certain of the transfectant clones. Here we demonstrate that over‐expression of B7‐1 can inhibit tumor development by certain AB1‐B7‐1 clones, that inhibition of transfectant growth is dependent on CD4+ and CD8+ T cells and that mice that reject some of these transfectant clones are capable of rejecting subsequent inocula of the parental cell line, AB1. The transfectant clones can generate tumor‐specific cytotoxic T cells. By contrast, expression of B7‐2 in several clones derived from either AB1 or AC29 had no significant effect on the development of tumors in vivo. Our data are consistent with data from other systems that show differences in the effect of modification by B7‐1 or B7‐2 on the modulation of anti‐tumor immune responses. They demonstrate that such modifications can induce protective immunity against an MM cell line but confirm the intra‐ and inter‐tumoral heterogeneity in the effect of genetic modification on the induction of immunity. Our observations are relevant to human MM because these cell lines have been derived from asbestos‐induced tumors and share many properties with human cell lines of the same histological type. Int. J. Cancer 71:476‐482, 1997.

Microsatellite, restriction fragment-length polymorphism, and sequence-specific oligonucleotide typing of the tumor necrosis factor region comparisons of the 4AOHW cell panel☆

The location of the TNF and other genes in the central MHC and their possible relevance to disease susceptibility provided an impetus to develop useful typing markers. The 4AOHW undertook to assess the various markers available, including DNA sequence-based systems. A panel of well-characterized lymphoblastoid cell lines were typed by Nco I RLFP analysis, SSO typing, and TNF microsatellite typing. RFLP and SSO typing were relatively reproducible as judged by the blind replicates. The two techniques provided the same results with only one exception, and it would be reasonable to prefer SSO typing because of its advantages in terms of cost and time. Microsatellite typing was much more discriminating but, as expected, less robust in that some discrepancies were apparent. As a result of the workshop and subsequent testing, alleles and haplotypes were allocated to most cells within the 4AOHW panel, including 10W cells typed in previous studies. While there was evidence that microsatellites may be relatively stable, they have the potential to identify recent mutations within ancestral haplotypes.

Quality indicators of diabetes care: an example of remote-area Aboriginal primary health care over 10 years

Objectives: To describe service characteristics of Derby Aboriginal Health Service (DAHS) and document diabetes management activities and intermediate clinical outcomes for Aboriginal patients with type 2 diabetes.

The induction of immune responses to murine malignant mesothelioma by IL-2 gene transfer

Stable IL‐2 transfectant clones have been derived from two non‐immunogenic murine malignant mesothelioma (MM) cell lines to investigate the induction of protective antitumour immunity to MM. AC29‐IL‐2 transfectant clones grew at a slower rate in vivo than the parental cell line or a transfectant control clone but all inoculated mice developed tumours despite the continued ability of the tumour cells to express IL‐2. Tumour development after inoculation of AB1‐IL 2 transfectants varied, the degree of in vivo inhibition (40–100%) being directly related to the rate of IL‐2 secretion of the transfectants. When mice which had rejected the ABl‐lL‐2 transfectants were challenged with parental AB1 cells, a proportion (16–70%) of mice from each group remained tumour free at least 45 days after challenge (naive mice developed tumours within 26 days). The inhibition of growth of the initial inoculum of ABl‐IL‐2 transfectants was independent of CD4+ and CD8+ cells, consistent with the demonstration of non‐specific cytotoxic activity by splenocytes from mice inoculated with the IL‐2 transfectants. These data suggest that IL‐2 expression by MM cells is capable of generating in vivo immunity to the tumour. This immunity may be relatively weak or may be subject to downregulation so that consistent rejection of unmodified tumour cells is not achieved. Genetic modification with combinations of genes, including lL‐2 and B7‐1.will be necessary for reliable generation of protective immunity to MM.

Cross-sectional comparison of point-of-care with laboratory HbA1c in detecting diabetes in real-world remote Aboriginal settings

Objectives To determine if point-of-care (POC) glycated haemoglobin (HbA1c) is sufficiently accurate in real-world remote settings to predict or exclude the diagnosis of diabetes based on laboratory HbA1c measurements. Design Cross-sectional study comparing POC capillary HbA1c results with corresponding venous HbA1c levels measured in a reference laboratory. Participants Aboriginal patients ≥15 years old who were due for diabetes screening at the participating clinics were invited to participate. Two hundred and fifty-five Aboriginal participants were enrolled and 241 were included in the analysis. Setting 6 primary healthcare sites in the remote Kimberley region of Western Australia from September 2011 to November 2013. Main outcome measures Concordance and mean differences between POC capillary blood HbA1c measurement and laboratory measurement of venous blood HbA1c level; POC capillary blood HbA1c equivalence value for screening for diabetes or a high risk of developing diabetes; sensitivity, specificity and positive-predictive value for diagnosing and screening for diabetes; barriers to conducting POC testing. Results Concordance between POC and laboratory results was good (ρ=0.88, p<0.001). The mean difference was −0.15% (95% limits of agreement, −0.67% to 0.36%). POC HbA1c measurements ≥6.5%, 48 mmol/mol had a specificity of 98.2% and sensitivity of 73.7% for laboratory measurements ≥6.5%. The POC equivalence value for screening for diabetes or a high risk of developing diabetes was ≥5.7%, 39 mmol/mol (sensitivity, 91%; specificity, 76.7% for laboratory measurements ≥6.0%, 42 mmol/mol). Staff trained by other clinic staff ‘on the job’ performed as well as people with formal accredited training. Staff reported difficulty in maintaining formal accreditation. Conclusions POC HbA1c testing is sufficiently accurate to be a useful component in screening for, and diagnosing, diabetes in remote communities. Limited local training is adequate to produce results comparable to laboratory results and accreditation processes need to reflect this.

Clinical trials in a remote Aboriginal setting: Lessons from the BOABS smoking cessation study

BackgroundThere is limited evidence regarding the best approaches to helping Indigenous Australians to stop smoking. The composite analysis of the only two smoking cessation randomised controlled trials (RCTs) investigating this suggests that one-on-one extra support delivered by and provided to Indigenous Australians in a primary health care setting appears to be more effective than usual care in encouraging smoking cessation. This paper describes the lessons learnt from one of these studies, the Be Our Ally Beat Smoking (BOABS) Study, and how to develop and implement an integrated smoking cessation program.MethodsQualitative study using data collected from multiple documentary sources related to the BOABS Study. As the project neared completion the research team participated in four workshops to review and conduct thematic analyses of these documents.ResultsChallenges we encountered during the relatively complex BOABS Study included recruiting sufficient number of participants; managing the project in two distant locations and ensuring high quality work across both sites; providing appropriate training and support to Aboriginal researchers; significant staff absences, staff shortages and high workforce turnover; determining where and how the project fitted in the clinics and consequent siloing of the Aboriginal researchers relating to the requirements of RCTs; resistance to change, and maintaining organisational commitment and priority for the project. The results of this study also demonstrated the importance of local Aboriginal ownership, commitment, participation and control. This included knowledge of local communities, the flexibility to adapt interventions to local settings and circumstances, and taking sufficient time to allow this to occur.ConclusionsThe keys to the success of the BOABS Study were local development, ownership and participation, worker professional development and support, and operating within a framework of cultural safety. There were difficulties associated with the BOABS Study being an RCT, and many of these are shared with stand-alone programs. Interventions targeted at particular health problems are best integrated with usual primary health care. Research to investigate complex interventions in Indigenous health should not be limited to randomised clinical trials and funding needs to reflect the additional, but necessary, cost of providing for local control of planning and implementation.

Baseline investigations of folate status in Aboriginal and non-Aboriginal West Australians prior to the introduction of mandatory fortification

In September 2009, Australia implemented mandatory folic acid fortification of wheat flour for bread‐making to reduce the incidence of neural tube defects. Our study aimed to establish baseline folate status data in Aboriginal and non‐Aboriginal Western Australians.

Aboriginal health research in the remote Kimberley: an exploration of perceptions, attitudes and concerns of stakeholders

BackgroundFor decades Indigenous peoples have argued for health research reform claiming methods used and results obtained often reflect the exploitative history of colonisation. In 2006 the Kimberley Aboriginal Health Planning Forum (KAHPF) Research Subcommittee (hereafter, the Subcommittee) was formed to improve research processes in the remote Kimberley region of north Western Australia. This paper explores the major perceptions, attitudes and concerns of stakeholders in the Subcommittee.MethodsQualitative analysis was carried out on data retrospectively collected from multiple evidentiary sources linked to the Subcommittee i.e. database, documents, interviews, review forms and emails from 1 January 2007 to 31 October 2013.ResultsFrom 1 January 2007 to 30 June 2013 the Subcommittee received 95 proposals, 57 (60%) driven by researchers based outside the region. Local stakeholders (22 from 12 different Kimberley organisations) raised concerns about 36 (38%) projects, 30 (83%) of which were driven by external researchers. Major concerns of local stakeholders were inadequate community consultation and engagement; burden of research on the region; negative impact of research practices; lack of demonstrable community benefit; and power and control of research. Major themes identified by external stakeholders (25 external researchers who completed the review form) were unanticipated difficulties with consultation processes; barriers to travel; perceiving research as a competing priority for health services and time-consuming ethics processes. External stakeholders also identified strategies for improving research practices in the Kimberley: importance of community support in building good relationships; employing local people; flexibility in research approaches; and importance of allocating sufficient time for consultation and data collection.ConclusionsHealth research in the Kimberley has improved in recent years, however significant problems remain. Prioritising research addressing genuine local needs is essential in closing the gap in Indigenous life expectancy. The long-term aim is for local health service connected researchers to identify priorities, lead, conduct and participate in the majority of local health research. For this to occur, a more radical move involving reconceptualising the research process is needed. Changes to institutional timeframes and funding processes could improve Indigenous and community-based research.