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1.  CD45 ligation expands Tregs by promoting interactions with DCs 
The Journal of Clinical Investigation  2014;124(10):4603-4613.
Regulatory T cells (Tregs), which express CD4 and FOXP3, are critical for modulating the immune response and promoting immune tolerance. Consequently, methods to expand Tregs for therapeutic use are of great interest. While transfer of Tregs after massive ex vivo expansion can be achieved, in vivo expansion of Tregs would be more practical. Here, we demonstrate that targeting the CD45 tyrosine phosphatase with a tolerogenic anti-CD45RB mAb acutely increases Treg numbers in WT mice, even in absence of exogenous antigen. Treg expansion occurred through substantial augmentation of homeostatic proliferation in the preexisting Treg population. Moreover, anti-CD45RB specifically increased Treg proliferation in response to cognate antigen. Compared with conventional T cells, Tregs differentially regulate their conjugation with DCs. Therefore, we determined whether CD45 ligation could alter interactions between Tregs and DCs. Live imaging showed that CD45 ligation specifically reduced Treg motility in an integrin-dependent manner, resulting in enhanced interactions between Tregs and DCs in vivo. Increased conjugate formation, in turn, augmented nuclear translocation of nuclear factor of activated T cells (NFAT) and Treg proliferation. Together, these results demonstrate that Treg peripheral homeostasis can be specifically modulated in vivo to promote Treg expansion and tolerance by increasing conjugation between Tregs and DCs.
PMCID: PMC4191025  PMID: 25202978
2.  Systems Biology Approach to Transplant Tolerance: Proof of concept experiments using RNA Interference (RNAi) to knock down Hub Genes in Jurkat and HeLa Cells in vitro 
The Journal of surgical research  2011;176(1):e41-e46.
Systems biology is gaining importance in studying complex systems such as the functional interconnections of human genes. To investigate the molecular interactions involved in T cell immune responses, we used databases of physical gene-gene interactions to constructed molecular interaction networks (interconnections) with R language algorithms. This helped to identify highly interconnected “hub” genes AT(1)P5C1, IL6ST, PRKCZ, MYC, FOS, JUN and MAPK1. We hypothesized that suppression of these hub genes in the gene network would result in significant phenotypic effects on T cells and examined this in-vitro. The molecular interaction networks were then analyzed and visualized with Cytoscape.
Materials and Methods
Jurkat and HeLa cells were transfected with siRNA for the selected hub genes. Cell proliferation was measured using ATP luminescence and BrdU labeling, which were measured 36, 72 and 96 hours after activation.
Following T cell stimulation, we found a significant decrease in ATP production (P<0.05) when the hub genes ATP5C1 and PRKCZ were knocked down using siRNA transfection, whereas no difference in ATP production was observed in siRNA transfected HeLa cells. However, HeLa cells showed a significant (P<0.05) decrease in cell proliferation when the genes MAPK1, IL6ST, ATP5C1, JUN and FOS were knocked down.
In both Jukat and HeLa cells, targeted gene knockdown using siRNA showed decreased cell proliferation and ATP production in both Jurkat andHeLa cells. However, Jurkat T cells and HELA cells use different hub genes to regulate activation responses. This experiment provides proof of principle of applying siRNA knockdown of T cell hub genes to evaluate their proliferative capacity and ATP production. This novel concept outlines a systems biology approach to identify hub genes for targeted therapeutics.
PMCID: PMC4233152  PMID: 22342379
siRNA Jurkat cells; knockdown; hub genes; systems biology; transplant tolerance
3.  Self-reported contacts for mental health problems by rural residents: predicted service needs, facilitators and barriers 
BMC Psychiatry  2014;14(1):249.
Rural and remote Australians face a range of barriers to mental health care, potentially limiting the extent to which current services and support networks may provide assistance. This paper examines self-reported mental health problems and contacts during the last 12 months, and explores cross-sectional associations between potential facilitators/barriers and professional and non-professional help-seeking, while taking into account expected associations with socio-demographic and health-related factors.
During the 3-year follow-up of the Australian Rural Mental Health Study (ARMHS) a self-report survey was completed by adult rural residents (N = 1,231; 61% female; 77% married; 22% remote location; mean age = 59 years), which examined socio-demographic characteristics, current health status factors, predicted service needs, self-reported professional and non-professional contacts for mental health problems in the last 12 months, other aspects of help-seeking, and perceived barriers.
Professional contacts for mental health problems were reported by 18% of the sample (including 14% reporting General Practitioner contacts), while non-professional contacts were reported by 16% (including 14% reporting discussions with family/friends). Perceived barriers to health care fell under the domains of structural (e.g., costs, distance), attitudinal (e.g., stigma concerns, confidentiality), and time commitments. Participants with 12-month mental health problems who reported their needs as met had the highest levels of service use. Hierarchical logistic regressions revealed a dose-response relationship between the level of predicted need and the likelihood of reporting professional and non-professional contacts, together with associations with socio-demographic characteristics (e.g., gender, relationships, and financial circumstances), suicidal ideation, and attitudinal factors, but not geographical remoteness.
Rates of self-reported mental health problems were consistent with baseline findings, including higher rural contact rates with General Practitioners. Structural barriers displayed mixed associations with help-seeking, while attitudinal barriers were consistently associated with lower service contacts. Developing appropriate interventions that address perceptions of mental illness and attitudes towards help-seeking is likely to be vital in optimising treatment access and mental health outcomes in rural areas.
Electronic supplementary material
The online version of this article (doi:10.1186/s12888-014-0249-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4172961  PMID: 25193400
Rural; Mental health; Service utilisation; Treatment barriers; Attitudes
4.  Tocilizumab in the treatment of rheumatoid arthritis and beyond 
Rheumatoid arthritis (RA) is a systemic inflammatory disease characterized by joint pain, swelling, stiffness, and progressive destruction of the small joints of the hands and feet. Treatment of RA has improved over the past decade. With multiple cytokines well-known now to play a role in the pathogenesis of RA, including tumor necrosis factor alpha, interleukin (IL)-1β, and IL-6, many targeted biological treatments against these cytokines have emerged, changing the treatment of this disease. Tocilizumab (TCZ) is a recombinant humanized monoclonal antibody against the IL-6 receptor and has been approved in many countries, including the United States, for the treatment of moderate to severe RA in patients who have not adequately responded to one or more disease-modifying antirheumatic drugs (DMARDs) or cannot tolerate other approved drug classes for RA. The aim of this review is to discuss the role of IL-6 in RA, and to provide an overview of the mode of action, pharmacokinetics, and safety of TCZ. Furthermore, efficacy studies of TCZ as both monotherapy and combination therapy will be evaluated. There have been several important clinical trials evaluating the efficacy and safety of TCZ in RA patients; this review summarizes this data from 14 key trials with emphasis on Phase III trials. Review of these trials provides strong evidence that its use, both as monotherapy and in combination with methotrexate or other DMARDs, is an effective treatment in reducing the signs and symptoms of RA. TCZ showed tolerable safety but care is required for its use since there are some important safety concerns including elevated liver enzymes, elevated low-density lipoprotein, infections, and gastrointestinal perforations. Additionally, given the efficacy of TCZ in the treatment of RA, this review discusses how TCZ may be beneficial in the treatment of other autoimmune diseases, spinal disease, cardiovascular disease, organ transplantation, and malignancies where elevated levels of IL-6 may play a role in the pathogenesis of these diseases.
PMCID: PMC3974690  PMID: 24729685
tocilizumab; IL-6; rheumatoid arthritis; biologics
5.  Regulation of Cytotoxic T Lymphocyte Antigen 4 by Cyclic AMP 
Recent studies indicate that cyclic AMP (cAMP) induces cytotoxic T lymphocyte antigen (CTLA) 4. CTLA4 is expressed in T cells, and is a negative regulator of T cell activation. CTLA4 expression is regulated by T cell receptor plus CD28 (adaptive immune signaling) at both the transcriptional and post-transcriptional level. Here, we examine the pathways by which cAMP regulates CTLA4 expression, focusing on transcriptional activation. Elevating intracellular cAMP levels by cell-permeable cAMP analogs, the adenylyl cyclase activator, forskolin, or phosphodiesterase inhibitors increases CTLA4 mRNA expression in EL4 murine T cells and primary CD4+ T cells. Activation of protein kinase A (using the protein kinase A–selective agonist, N6-phenyladenosine-cAMP), but not exchange proteins activated by cAMP (using the exchange proteins activated by cAMP–selective 8-pCPT-2Me-cAMP), increases CTLA4 promoter activity. Mutation constructs of the CTLA4 promoter uncover an enhancer binding site located within the −150 to −130 bp region relative to the transcription start site. Promoter analysis and chromatin immunoprecipitation assays suggest that cAMP response element–binding is a putative transcription factor induced by cAMP. We have previously shown that CTLA4 mediates decreased pulmonary inflammation in an LPS-induced murine model of acute lung injury (ALI). We observed that LPS can induce CTLA4 transcription via the same cAMP-inducible promoter region. The immunosuppressant, rapamycin, decreases cAMP and LPS-induced CTLA4 transcription in vitro. In vivo, LPS induces cAMP accumulation in bronchoalveolar lavage fluid, bronchoalveolar lavage cells, and lung tissues in ALI. We demonstrate that rapamycin decreases cAMP accumulation and CTLA4 expression in ALI. Together, these data suggest that cAMP may negatively regulate pulmonary inflammatory responses in vivo and in vitro by altering CTLA4 expression.
PMCID: PMC3547085  PMID: 23024062
cytotoxic T lymphocyte antigen 4; cAMP; acute lung injury; protein kinase A; rapamycin
6.  Factors associated with reported service use for mental health problems by residents of rural and remote communities: cross-sectional findings from a baseline survey 
The patterns of health service use by rural and remote residents are poorly understood and under-represented in national surveys. This paper examines professional and non-professional service use for mental health problems in rural and remote communities in Australia.
A stratified random sample of adults was drawn from non-metropolitan regions of New South Wales, Australia as part of a longitudinal population-based cohort. One-quarter (27.7%) of the respondents were from remote or very remote regions. The socio-demographic, health status and service utilization (professional and non-professional) characteristics of 2150 community dwelling residents are described. Hierarchical logistic regressions were used to identify cross-sectional associations between socio-demographic, health status and professional and non-professional health service utilization variables.
The overall rate of professional contacts for mental health problems during the previous 12 months (17%) in this rural population exceeded the national rate (11.9%). Rates for psychologists and psychiatrists were similar but rates for GPs were higher (12% vs. 8.1%). Non-professional contact rates were 12%. Higher levels of help seeking were associated with the absence of a partner, poorer finances, severity of mental health problems, and higher levels of adversity. Remoteness was associated with lower utilization of non-professional support. A Provisional Service Need Index was devised, and it demonstrated a broad dose–response relationship between severity of mental health problems and the likelihood of seeking any professional or non-professional help. Nevertheless, 47% of those with estimated high service need had no contact with professional services.
An examination of self-reported patterns of professional and non-professional service use for mental health problems in a rural community cohort revealed relatively higher rates of general practitioner attendance for such problems compared with data from metropolitan centres. Using a measure of Provisional Service Need those with greater needs were more likely to access specialist services, even in remote regions, although a substantial proportion of those with the highest service need sought no professional help. Geographic and financial barriers to service use were identified and perception of service adequacy was relatively low, especially among those with the highest levels of distress and greatest adversity.
PMCID: PMC3655863  PMID: 23631501
Health service utilisation; Mental health; Rural health
7.  Individual and district-level predictors of alcohol use: cross sectional findings from a rural mental health survey in Australia 
BMC Public Health  2012;12:586.
Excessive alcohol use is a significant problem in rural and remote Australia. The factors contributing to patterns of alcohol use have not been adequately explained, yet the geographic variation in rates suggests a potential contribution of district-level factors, such as socio-economic disadvantage, rates of population change, environmental adversity, and remoteness from services/population centres. This paper aims to investigate individual-level and district-level predictors of alcohol use in a sample of rural adults.
Using baseline survey data (N = 1,981) from the population-based Australian Rural Mental Health Study of community dwelling residents randomly selected from the Australia electoral roll, hierarchal logistic regression models were fitted for three outcomes: 1) at-risk alcohol use, indicated by Alcohol Use Disorders Identification Test scores ≥8; 2) high alcohol consumption (> 40 drinks per month); and 3) lifetime consequences of alcohol use. Predictor variables included demographic factors, pre-dispositional factors, recent difficulties and support, mental health, rural exposure and district-level contextual factors.
Gender, age, marital status, and personality made the largest contribution to at-risk alcohol use. Five or more adverse life events in the past 12 months were also independently associated with at-risk alcohol use (Adjusted Odds Ratio [AOR] 3.3, 99%CI 1.2, 8.9). When these individual-level factors were controlled for, at-risk alcohol use was associated with having spent a lower proportion of time living in a rural district (AOR 1.7, 99%CI 1.3, 2.9). Higher alcohol consumption per month was associated with higher district-level socio-economic ranking, indicating less disadvantage (AOR 1.2, 99%CI 1.02, 1.4). Rural exposure and district-level contextual factors were not significantly associated with lifetime consequences of alcohol use.
Although recent attention has been directed towards the potential adverse health effects of district or community level adversity across rural regions, our study found relatively few district-level factors contributing to at-risk alcohol consumption after controlling for individual-level factors. Population-based prevention strategies may be most beneficial in rural areas with a higher socio-economic ranking, while individual attention should be focused towards rural residents with multiple recent adverse life events, and people who have spent less time residing in a rural area.
PMCID: PMC3491021  PMID: 22853803
Alcohol; Mental health; Rural health
8.  The endogenous pro-resolving mediators lipoxin A4 and resolvin E1 preserve organ function in allograft rejection 
Allograft rejection remains a major limitation to successful solid organ transplantation. Here, we investigated the biosynthesis and bioactions of the pro-resolving mediators lipoxin A4 and resolvin E1 in host responses to organ transplantation. In samples obtained during screening bronchoscopy after human lung transplantation, bronchoalveolar lavage fluid levels of lipoxin A4 were increased in association with the severity of allograft rejection that was graded independently by clinical pathology. Lipoxin A4 significantly inhibited calcineurin activation in human neutrophils, and lipoxin A4 stable analogs prevented acute rejection of vascularized cardiac and renal allografts. Transgenic animals expressing human lipoxin A4 receptors revealed important sites of action in host tissues for lipoxin A4’s protective effects. Resolvin E1 displays counter-regulatory actions for leukocytes, in part, via increased lipoxin A4 biosynthesis, yet RvE1 administered (1 µg, iv) to donor (days −1 and 0) and recipient mice (day −1, 0 and +4) was even more potent than a lipoxin stable analog (1 µg, iv) in prolonging renal allograft survival (median survival time = 74.0 days with RvE1 and 37.5 days with a LXA4 analog). Together, these results highlight the potential for pro-resolving mediators in prolonging survival of solid organ transplants.
PMCID: PMC3019284  PMID: 20869861
9.  Shared care in mental illness: A rapid review to inform implementation 
While integrated primary healthcare for the management of depression has been well researched, appropriate models of primary care for people with severe and persistent psychotic disorders are poorly understood. In 2010 the NSW (Australia) Health Department commissioned a review of the evidence on "shared care" models of ambulatory mental health services. This focussed on critical factors in the implementation of these models in clinical practice, with a view to providing policy direction. The review excluded evidence about dementia, substance use and personality disorders.
A rapid review involving a search for systematic reviews on The Cochrane Database of Systematic Reviews and Database of Abstracts of Reviews of Effects (DARE). This was followed by a search for papers published since these systematic reviews on Medline and supplemented by limited iterative searching from reference lists.
Shared care trials report improved mental and physical health outcomes in some clinical settings with improved social function, self management skills, service acceptability and reduced hospitalisation. Other benefits include improved access to specialist care, better engagement with and acceptability of mental health services. Limited economic evaluation shows significant set up costs, reduced patient costs and service savings often realised by other providers. Nevertheless these findings are not evident across all clinical groups. Gains require substantial cross-organisational commitment, carefully designed and consistently delivered interventions, with attention to staff selection, training and supervision. Effective models incorporated linkages across various service levels, clinical monitoring within agreed treatment protocols, improved continuity and comprehensiveness of services.
"Shared Care" models of mental health service delivery require attention to multiple levels (from organisational to individual clinicians), and complex service re-design. Re-evaluation of the roles of specialist mental health staff is a critical requirement. As expected, no one model of "shared" care fits diverse clinical groups. On the basis of the available evidence, we recommended a local trial that examined the process of implementation of core principles of shared care within primary care and specialist mental health clinical services.
PMCID: PMC3235059  PMID: 22104323
10.  Fenfluramine-induced gene dysregulation in human pulmonary artery smooth muscle and endothelial cells 
Pulmonary Circulation  2011;1(3):405-418.
Fenfluramine is prescribed either alone or in combination with phentermine as part of Fen-Phen, an anti-obesity medication. Fenfluramine was withdrawn from the US market in 1997 due to reports of heart valvular disease, pulmonary arterial hypertension, and cardiac fibrosis. Particularly, idiopathic pulmonary arterial hypertension (IPAH), previously referred to as primary pulmonary hypertension (PPH), was found to be associated with the use of Fen-Phen, fenfluramine, and fenfluramine derivatives. The underlying mechanism of fenfluramine-associated pulmonary hypertension is still largely unknown. We reasoned that investigating drug-induced gene dysregulation would enhance our understanding of the fenfluramine-associated pathogenic mechanism of IPAH. Whole-genome gene expression profiles in fenfluramine-treated human pulmonary artery smooth muscle (PASMC) and endothelial (PAEC) cells (isolated from normal subjects) were compared with baseline expression in untreated cells. Fenfluramine treatment caused dysregulation in a substantial number of genes involved in a variety of pathways and biological processes. In addition to several common pathways and biological processes such as “MAPK signaling pathway,” “inflammation response,” and “calcium signaling pathway” shared between both cell types, pathways and biological processes such as “blood circulation,” “muscle system process,” and “immune response” were enriched among the dysregulated genes in PASMC. Pathways and biological processes such as those related to cell cycle, however, were enriched among the dysregulated genes in PAEC, indicating that fenfluramine could affect unique pathways (or differentially) in different types of pulmonary artery cells. While awaiting validation in a larger cohort, these results strongly suggested that fenfluramine could induce significant dysregulation of genes in multiple biological processes and pathways critical for normal pulmonary vascular functions and structure. The transcriptional and posttranscriptional changes in these genes may, therefore, contribute to the pathogenesis of fenfluramine-associated IPAH.
PMCID: PMC3224433  PMID: 22140631
anorexigen; gene expression profile; lysosome; mitochondria; pulmonary hypertension
11.  Surfactant Protein D Mediated Decrease of Allergen-Induced Inflammation is Dependent upon CTLA41 
Pulmonary surfactant protein D (SP-D), a member of the collectin family, is an innate immune molecule critical for defense that can also modulate adaptive immune responses. We previously showed that SP-D deficient mice exhibit enhanced allergic responses, and that SP-D induction requires lymphocytes. Thus, we postulated that SP-D may decrease adaptive allergic responses through interaction with T cells. In this study, we used two forms of SP-D, a dodecamer (SP-D dodec) and a shorter fragment containing the trimeric neck and carbohydrate recognition domains (SP-D NCRD). Both forms decreased immune responses in vitro and in a murine model of pulmonary inflammation. SP-D NCRD increased transcription of cytotoxic T lymphocyte antigen 4 (CTLA4), a negative regulator of T cell activation, in T cells. SP-D NCRD no longer decreased lymphoproliferation and IL-2 cytokine production when CTLA4 signals were abrogated. Administration of SP-D NCRD in vivo no longer decreased allergen induced responses when CTLA4 was inhibited. Our results indicate that SP-D decreases allergen responses, an effect that may be mediated by increase of CTLA4 in T cells.
PMCID: PMC2905687  PMID: 20435925
SP-D; CTLA4; allergy; inflammation
12.  T cell pathways involving CTLA4 contribute to a model of acute lung injury1 
Acute lung injury (ALI) is a frequent pulmonary complication in critically ill patients. We characterized a murine model of LPS-induced ALI, focusing on T helper cells. Following LPS administration, BAL lymphocytes were increased as well as neutrophils, IL-6, TNF-α, and albumin. Analysis of LPS-induced T cells revealed increased T helper cell associated cytokines (IL-17A, IL-17F, and IL-22), expression of CD69, a cell activation marker, forkhead box P3 (Foxp3), and cytotoxic T lymphocyte antigen 4 (CTLA4) in CD4+ T cells. Administration of anti-CTLA4 antibody decreased LPS-induced BAL albumin and IL-17A, while increasing CD4+Foxp3+ cell number and Foxp3 expression in CD4+Foxp3+ cells. These data suggest that pulmonary LPS administration promotes CD4+ T cells and that T cell pathways involving CTLA4 contribute to ALI.
PMCID: PMC3068917  PMID: 20385880
T cells; CTLA4; LPS; acute lung injury
13.  Effectiveness of service linkages in primary mental health care: a narrative review part 1 
With the move to community care and increased involvement of generalist health care providers in mental health, the need for health service partnerships has been emphasised in mental health policy. Within existing health system structures the active strategies that facilitate effective partnership linkages are not clear. The objective of this study was to examine the evidence from peer reviewed literature regarding the effectiveness of service linkages in primary mental health care.
A narrative and thematic review of English language papers published between 1998 and 2009. Studies of analytic, descriptive and qualitative designs from Australia, New Zealand, UK, Europe, USA and Canada were included. Data were extracted to examine what service linkages have been used in studies of collaboration in primary mental health care. Findings from the randomised trials were tabulated to show the proportion that demonstrated clinical, service delivery and economic benefits.
A review of 119 studies found ten linkage types. Most studies used a combination of linkage types and so the 42 RCTs were grouped into four broad linkage categories for meaningful descriptive analysis of outcomes. Studies that used multiple linkage strategies from the suite of "direct collaborative activities" plus "agreed guidelines" plus "communication systems" showed positive clinical (81%), service (78%) and economic (75%) outcomes. Most evidence of effectiveness came from studies of depression. Long term benefits were attributed to medication concordance and the use of case managers with a professional background who received expert supervision. There were fewer randomised trials related to collaborative care of people with psychosis and there were almost none related to collaboration with the wider human service sectors. Because of the variability of study types we did not exclude on quality or attempt to weight findings according to power or effect size.
There is strong evidence to support collaborative primary mental health care for people with depression when linkages involve "direct collaborative activity", plus "agreed guidelines" and "communication systems".
PMCID: PMC3079614  PMID: 21481236
Narrative review; mental health services; primary health care; cooperative behaviour
14.  Building effective service linkages in primary mental health care: a narrative review part 2 
Primary care services have not generally been effective in meeting mental health care needs. There is evidence that collaboration between primary care and specialist mental health services can improve clinical and organisational outcomes. It is not clear however what factors enable or hinder effective collaboration. The objective of this study was to examine the factors that enable effective collaboration between specialist mental health services and primary mental health care.
A narrative and thematic review of English language papers published between 1998 and 2009. An expert reference group helped formulate strategies for policy makers. Studies of descriptive and qualitative design from Australia, New Zealand, UK, Europe, USA and Canada were included. Data were extracted on factors reported as enablers or barriers to development of service linkages. These were tabulated by theme at clinical and organisational levels and the inter-relationship between themes was explored.
A thematic analysis of 30 papers found the most frequently cited group of factors was "partnership formation", specifically role clarity between health care workers. Other factor groups supporting clinical partnership formation were staff support, clinician attributes, clinic physical features and evaluation and feedback. At the organisational level a supportive institutional environment of leadership and change management was important. The expert reference group then proposed strategies for collaboration that would be seen as important, acceptable and feasible. Because of the variability of study types we did not exclude on quality and findings are weighted by the number of studies. Variability in local service contexts limits the generalisation of findings.
The findings provide a framework for health planners to develop effective service linkages in primary mental health care. Our expert reference group proposed five areas of strategy for policy makers that address organisational level support, joint clinical problem solving, local joint care guidelines, staff training and supervision and feedback.
PMCID: PMC3070626  PMID: 21435273
Narrative review; mental health services; primary health care; cooperative behaviour
15.  Advances of genomic science and systems biology in renal transplantation: a review 
Seminars in Immunopathology  2011;33(2):211-218.
The diagnosis of rejection in kidney transplant patients is based on histologic classification of a graft biopsy. The current “gold standard” is the Banff 97 criteria; however, there are several limitations in classifying rejection based on biopsy samples. First, a biopsy involves an invasive procedure. Second, there is significant variance among blinded pathologists in the interpretation of a biopsy. And third, there is also variance between the histology and the molecular profiles of a biopsy. To increase the positive predictive value of classifiers of rejection, a Banff committee is developing criteria that integrate histologic and molecular data into a unified classifier that could diagnose and prognose rejection. To develop the most appropriate molecular criteria, there have been studies by multiple groups applying omics technologies in attempts to identify biomarkers of rejection. In this review, we discuss studies using genome-wide data sets of the transcriptome and proteome to investigate acute rejection, chronic allograft dysfunction, and tolerance. We also discuss studies which focus on genetic biomarkers in urine and peripheral blood, which will provide clinicians with minimally invasive methods for monitoring transplant patients. We also discuss emerging technologies, including whole-exome sequencing and RNA-Seq and new bioinformatic and systems biology approaches, which should increase the ability to develop both biomarkers and mechanistic understanding of the rejection process.
PMCID: PMC3082700  PMID: 21318414
Kidney transplantation; Acute rejection; Chronic rejection; Rna-seq; Genomics; Microarrays; Modulated genes
16.  Integrated primary health care in Australia 
To fulfil its role of coordinating health care, primary health care needs to be well integrated, internally and with other health and related services. In Australia, primary health care services are divided between public and private sectors, are responsible to different levels of government and work under a variety of funding arrangements, with no overarching policy to provide a common frame of reference for their activities.
Description of policy
Over the past decade, coordination of service provision has been improved by changes to the funding of private medical and allied health services for chronic conditions, by the development in some states of voluntary networks of services and by local initiatives, although these have had little impact on coordination of planning. Integrated primary health care centres are being established nationally and in some states, but these are too recent for their impact to be assessed. Reforms being considered by the federal government include bringing primary health care under one level of government with a national primary health care policy, establishing regional organisations to coordinate health planning, trialling voluntary registration of patients with general practices and reforming funding systems. If adopted, these could greatly improve integration within primary health care.
Careful change management and realistic expectations will be needed. Also other challenges remain, in particular the need for developing a more population and community oriented primary health care.
PMCID: PMC2787230  PMID: 19956377
primary health care; health policy; integration; Australia
17.  What evidence is there to support skill mix changes between GPs, pharmacists and practice nurses in the care of elderly people living in the community? 
Workforce shortages in Australia are occurring across a range of health disciplines but are most acute in general practice. Skill mix change such as task substitution is one solution to workforce shortages. The aim of this systematic review was to explore the evidence for the effectiveness of task substitution between GPs and pharmacists and GPs and nurses for the care of older people with chronic disease. Published, peer reviewed (black) and non-peer reviewed (grey) literature were included in the review if they met the inclusion criteria.
Forty-six articles were included in the review. Task substitution between pharmacists and GPs and nurses and GPs resulted in an improved process of care and patient outcomes, such as improved disease control. The interventions were either health promotion or disease management according to guidelines or use of protocols, or a mixture of both. The results of this review indicate that pharmacists and nurses can effectively provide disease management and/or health promotion for older people with chronic disease in primary care. While there were improvements in patient outcomes no reduction in health service use was evident.
When implementing skill mix changes such as task substitution it is important that the health professionals' roles are complementary otherwise they may simply duplicate the task performed by other health professionals. This has implications for the way in which multidisciplinary teams are organised in initiatives such as the GP Super Clinics.
PMCID: PMC2749853  PMID: 19744350
18.  Integrated multi-level quality control for proteomic profiling studies using mass spectrometry 
BMC Bioinformatics  2008;9:519.
Proteomic profiling using mass spectrometry (MS) is one of the most promising methods for the analysis of complex biological samples such as urine, serum and tissue for biomarker discovery. Such experiments are often conducted using MALDI-TOF (matrix-assisted laser desorption/ionisation time-of-flight) and SELDI-TOF (surface-enhanced laser desorption/ionisation time-of-flight) MS. Using such profiling methods it is possible to identify changes in protein expression that differentiate disease states and individual proteins or patterns that may be useful as potential biomarkers. However, the incorporation of quality control (QC) processes that allow the identification of low quality spectra reliably and hence allow the removal of such data before further analysis is often overlooked. In this paper we describe rigorous methods for the assessment of quality of spectral data. These procedures are presented in a user-friendly, web-based program. The data obtained post-QC is then examined using variance components analysis to quantify the amount of variance due to some of the factors in the experimental design.
Using data from a SELDI profiling study of serum from patients with different levels of renal function, we show how the algorithms described in this paper may be used to detect systematic variability within and between sample replicates, pooled samples and SELDI chips and spots. Manual inspection of those spectral data that were identified as being of poor quality confirmed the efficacy of the algorithms. Variance components analysis demonstrated the relatively small amount of technical variance attributable to day of profile generation and experimental array.
Using the techniques described in this paper it is possible to reliably detect poor quality data within proteomic profiling experiments undertaken by MS. The removal of these spectra at the initial stages of the analysis substantially improves the confidence of putative biomarker identification and allows inter-experimental comparisons to be carried out with greater confidence.
PMCID: PMC2657802  PMID: 19055809
19.  Engaging participants in a complex intervention trial in Australian General Practice 
The paper examines the key issues experienced in recruiting and retaining practice involvement in a large complex intervention trial in Australian General Practice.
Reflective notes made by research staff and telephone interviews with staff from general practices which expressed interest, took part or withdrew from a trial of a complex general practice intervention.
Recruitment and retention difficulties were due to factors inherent in the demands and context of general practice, the degree of engagement of primary care organisations (Divisions of General Practice), perceived benefits by practices, the design of the trial and the timing and complexity of data collection.
There needs to be clearer articulation to practices of the benefits of the research to participants and streamlining of the design and processes of data collection and intervention to fit in with their work practices. Ultimately deeper engagement may require additional funding and ongoing participation through practice research networks.
Trial Registration
Current Controlled Trials ACTRN12605000788673
PMCID: PMC2533668  PMID: 18700984
20.  Contextual Control by Function and form of Transfer of Functions 
This study investigated conditions leading to contextual control by stimulus topography over transfer of functions. Three 4-member stimulus equivalence classes, each consisting of four (A, B, C, D) topographically distinct visual stimuli, were established for 5 college students. Across classes, designated A stimuli were open-ended linear figures, B stimuli were circular, C stimuli three-sided, and D stimuli four-sided. Three different computer tasks then were trained with the B stimuli. Differential reinforcement and punishment procedures were then used to establish control over function transfer by the topography of the class members. For Task 1, function transfer, responding to C and D stimuli as subjects had to B stimuli, was reinforced. For Task 2, function transfer was reinforced for C stimuli but punished for D stimuli. For Task 3, function transfer was punished for both C and D stimuli. New equivalence classes were then established and tests for generalized contextual control were presented. All 5 subjects showed generalized contextual control of transfer of functions by stimulus topography. Implications of contextual control over function transfer in natural settings are discussed.
PMCID: PMC1918086  PMID: 17725053
stimulus equivalence; transformation of function; transfer of function; contextual control; stimulus topography; button press; humans
21.  Prenatal, perinatal, and heritable influences on cord blood immune responses 
Maternal and perinatal environmental exposures, as well as inherited factors, may influence neonatal immune responses.
To determine relations of maternal and perinatal exposures to antigen-specific cord blood lymphoproliferative responses.
In 427 newborns from a Boston pregnancy/birth cohort, lymphoproliferative responses in cord blood mononuclear cells to stimulation with cockroach (Bla g 2), house dust mite (Der f 1), ovalbumin, and mitogen phytohemagglutinin were measured as stimulation index (SI). We used the Wilcoxon rank sum and χ2 tests to evaluate predictors of ovalbumin SI as a continuous ranked or dichotomous outcome. We used t test and Spearman correlation for univariate testing and linear regression to evaluate predictors of natural log-transformed Bla g 2, Der f 1, and phytohemagglutinin SI. Logistic multivariate regression was applied to evaluate predictors of Bla g 2, Der f 1, and phytohemagglutinin SI dichotomized at 2 or at the median for phytohemagglutinin.
Maternal smoking during pregnancy, inadequate or excessive maternal weight gain during pregnancy, neonate black race/ethnicity (compared with white), and Apgar score less than 8 were each independently associated with increased cord blood mononuclear cell proliferative responses to stimulation with Bla g 2 and/or Der f 1. Maternal history of asthma was associated only with increased lymphoproliferative response to ovalbumin stimulation.
Distinct fetal and perinatal exposures and black race/ethnicity may be associated with increased cord blood lymphoproliferative responses. The implications of these findings for future development of allergy or asthma are, as yet, unknown.
PMCID: PMC1562525  PMID: 16597079
22.  Associations of cord blood fatty acids with lymphocyte proliferation, IL-13, and IFN-γ 
Background. N-3 and n-6 polyunsaturated fatty acids (PUFAs) have been hypothesized to have opposing influences on neonatal immune responses that might influence the risk of allergy or asthma. However, both n-3 eicosapentaenoic acid (EPA) and n-6 arachidonic acid (AA) are required for normal fetal development.
Objective. We evaluated whether cord blood fatty acid levels were related to neonatal immune responses and whether n-3 and n-6 PUFA responses differed.
Methods. We examined the relation of cord blood plasma n-3 and n-6 PUFAs (n = 192) to antigen- and mitogen-stimulated cord blood lymphocyte proliferation (n = 191) and cytokine (IL-13 and IFN-γ; n = 167) secretion in a US birth cohort.
Results. Higher levels of n-6 linoleic acid were correlated with higher IL-13 levels in response to Bla g 2 (cockroach, P = .009) and Der f 1 (dust mite, P = .02). Higher n-3 EPA and n-6 AA levels were each correlated with reduced lymphocyte proliferation and IFN-γ levels in response to Bla g 2 and Der f 1 stimulation. Controlling for potential confounders, EPA and AA had similar independent effects on reduced allergen-stimulated IFN-γ levels. If neonates had either EPA or AA levels in the highest quartile, their Der f 1 IFN-γ levels were 90% lower (P = .0001) than those with both EPA and AA levels in the lowest 3 quartiles. Reduced AA/EPA ratio was associated with reduced allergen-stimulated IFN-γ level.
Conclusion. Increased levels of fetal n-3 EPA and n-6 AA might have similar effects on attenuation of cord blood lymphocyte proliferation and IFN-γ secretion.
Clinical implications. The implications of these findings for
PMCID: PMC1508138  PMID: 16630954
Asthma; child; cord blood; cytokine; fatty acids; lymphocyte proliferation; AA: Arachidonic acid; BMI: Body mass index; CBMC: Cord blood mononuclear cell; CI: Confidence interval; DHA: Docosohexaenoic acid; EPA: Eicosapentaenoic acid; FA: Fatty acid; LA: Linoleic acid; NICU: Neonatal intensive care unit; OVA: Ovalbumin; PG: Prostaglandin; PUFA: Polyunsaturated fatty acid; SI: Stimulation index
23.  Nuclear Factor Kappa B Activation in Human Cord Blood Mononuclear Cells 
Pediatric research  2004;56(2):212-218.
The immunologic signals participating in immune responses early in life have not been completely elucidated. Regarding the characterization of neonatal cells, little is known concerning the activity of transcription factor nuclear factor kappa B (NF-κB), which regulates inflammatory genes and cytokine production. The aim of this study was to characterize NF-κB activation in cord blood mononuclear cells (CBMC). We analyzed the potential association of NF-κB activity with lymphocyte proliferation and influences on cytokine secretion in the early immune system. To determine the contribution of a disease whereby inheritance may impact neonatal immunity, we assessed the influence of maternal allergic disease on NF-κB regulation and cytokine secretion. CBMC from healthy newborns were isolated and stimulated with mitogen (n = 28). Nuclear extracts were analyzed by electrophoretic mobility shift assay, cytokine secretion by ELISA. FISH analysis excluded relevant maternal contamination of CBMC. All samples showed a positive lymphoproliferative response, and NF-κB activity was both increased and decreased after mitogen stimulation. Increased NF-κB activation was significantly associated with decreased TNF-α secretion (median 6.1 versus 50.3 pg/mL) in unstimulated CBMC. Mitogen stimulation resulted in increased NF-κB activity with a trend to increased IL-13 production. Maternal allergic disease was associated with higher TNF-α (median 982 versus 173 pg/mL) and IL-13 secretion (median 1328 versus 1120 pg/mL) after mitogen stimulation. Together, NF-κB activity is differentially activated in cord blood and associated with a distinct cytokine pattern. Whether differential NF-κB activity in cord blood is related to the subsequent development of immune diseases requires further investigation.
PMCID: PMC1488728  PMID: 15181194
CBMC, cord blood mononuclear cells; EMSA, electrophoretic mobility shift assay; IFN-γ, interferon gamma; NF-κB, nuclear factor kappa B; PHA, phytohemagglutinin; SI, stimulation index; Th, T helper; TNF-α, tumor necrosis factor alpha
24.  (R)-albuterol decreases immune responses: role of activated T cells 
Respiratory Research  2008;9(1):3.
Racemic albuterol is an equimolar mixture of two isomers, (R) and (S). Whether (R) and (S) isomers and the combination of both exert different effects in immune activation is not well defined. We analyzed the effects of (R+S)-albuterol, (R)-albuterol and (S)-albuterol in a murine model of allergic pulmonary inflammation and in activated T cells. Mice (C57BL/6) sensitized and aerosol challenged with the allergen ovalbumin (OVA) or phosphate buffered saline (PBS) were treated with (R)-albuterol, (S)-albuterol or (R+S)-albuterol. Following administration of (R)-albuterol, allergen induced bronchoalveolar lavage eosinophils and IgE showed a decrease, albeit not significantly by ANOVA. As T cells are important in allergic inflammation, we asked whether (R+S), (R) or (S)-albuterol might differ in effects on T cells and on the activity of the inflammatory transcription factor NF-κB. In activated T cells, (R)-albuterol administration decreased levels of inflammatory cytokines and NF-κB activity. These studies suggest that (R)-albuterol decreases cytokine secretion and NF-κB activity in T cells.
PMCID: PMC2253534  PMID: 18194569
25.  TLR2 and TLR4 Stimulation Differentially Induce Cytokine Secretion in Human Neonatal, Adult, and Murine Mononuclear Cells 
Toll-like receptor 2 (TLR2) and TLR4 signaling may induce differential secretion of T helper 1 (Th1) and Th2 cytokines, potentially influencing the development of autoimmune or atopic diseases. To date, the influence of the type of stimulus, timing, and dose of TLR2 and TLR4 ligands on cytokine secretion has not been well established. We tested whether the innate stimuli peptidoglycan (Ppg, TLR2 agonist) and lipid A (LpA, TLR4 agonist) differentially affect the secretion of interleukin-13 (IL-13) (Th2) and interferon-γ (IFN-γ) (Th1). Further, we examined the influence of the maturity of the immune system, species, dose, and timing of stimuli in human cord and adult peripheral blood mononuclear cells (PBMC) and murine cells in vitro and in vivo. Stimulation with Ppg induced the secretion of both IL-13 and IFN-γ, influenced by time and dose in neonates, adults, and mice. In contrast, stimulation with LpA induced primarily time-independent and dose-independent production of IFN-γ. Pulmonary administration of Ppg in vivo in mice resulted in secretion of IL-13, whereas administration of LpA resulted in secretion of IFN-γ in bronchoalveolar lavage (BAL). Therefore, TLR2 and TLR4 stimuli differentially influence IL-13 and IFN-γ secretion in neonates, adults, and mice, supporting a critical role for innate stimuli in the modulation of cytokine responses.
PMCID: PMC2052924  PMID: 15450130

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