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Hughes A.,SA Pathology | Hughes A.,South Australia Health and Medical Research Institute | Hughes A.,University of Adelaide | Clarson J.,SA Pathology | And 11 more authors.
Blood | Year: 2017

Immunological control may contribute to achievement of deep molecular response in chronic myeloid leukemia (CML) patients on tyrosine kinase inhibitor (TKI) therapy and may promote treatment-free remission (TFR). We investigated effector and suppressor immune responses in CML patients at diagnosis (n = 21), on TKI (imatinib, nilotinib, dasatinib) before achieving major molecular response (pre-MMR, BCR-ABL1 >0.1%, n = 8), MMR (BCR-ABL1 ≤0.1%, n = 20), molecular response4.5 (MR4.5, BCR-ABL1 ≤0.0032%, n = 16), and sustained TFR (BCR-ABL1 undetectable following cessation of TKI therapy, n = 13). Aberrant immune-inhibitory responses (myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), and programmed death-1 (PD-1) inhibitory molecule expression on CD4+/CD8+ T cells were increased in CML patients at diagnosis. Consequent quantitative and functional defects of innate effector natural killer (NK) cells and cytotoxic T-lymphocyte responses to leukemia-associated antigens WT1, BMI-1, PR3, and PRAME were observed at diagnosis. Treg and PD-1+CD4+/CD8+ T cells persisted in pre-MMR CML patients on TKI. Patients in MMR and MR4.5 had a more mature, cytolytic CD57+CD62L- NK cell phenotype, consistent with restoration of NK cell activating and inhibitory receptor repertoire to normal healthy donor levels. Immune responses were retained in TFR patients off-therapy, suggesting the restored immune control observed in MMR and MR4.5 is not an entirely TKI-mediated effect. Maximal restoration of immune responses occurred only in MR4.5, as demonstrated by increased NK cell and effector T-cell cytolytic function, reduced T-cell PD-1 expression and reduced numbers of monocytic MDSCs. © 2017 by The American Society of Hematology.


Gargett T.,University of Adelaide | Gargett T.,Royal Adelaide Hospital | Grubor-Bauk B.,University of Adelaide | Garrod T.J.,University of Adelaide | And 6 more authors.
Immunology and Cell Biology | Year: 2014

The failure of traditional protein-based vaccines to prevent infection by viruses such as HIV or hepatitis C highlights the need for novel vaccine strategies. DNA vaccines have shown promise in small animal models, and are effective at generating anti-viral T cell-mediated immune responses; however, they have proved to be poorly immunogenic in clinical trials. We propose that the induction of necrosis will enhance the immune response to vaccine antigens encoded by DNA vaccines, as necrotic cells are known to release a range of intracellular factors that lead to dendritic cell (DC) activation and enhanced cross-presentation of antigen. Here we provide evidence that induction of cell death in DNA vaccine-targeted cells provides an adjuvant effect following intradermal vaccination of mice; however, this enhancement of the immune response is dependent on both the mechanism and timing of cell death after antigen expression. We report that a DNA vaccine encoding the cytolytic protein, perforin, resulted in DC activation, enhanced broad and multifunctional CD8 T-cell responses to the HIV-1 antigen GAG and reduced viral load following challenge with a chimeric virus, EcoHIV, compared with the canonical GAG DNA vaccine. This effect was not observed for a DNA vaccine encoding an apoptosis-inducing toxin, DTa, or when the level of perforin expression was increased to induce cell death sooner after vaccination. Thus, inducing lytic cell death following a threshold level of expression of a viral antigen can improve the immunogenicity of DNA vaccines, whereas apoptotic cell death has an inhibitory effect on the immune response. © 2014 Australasian Society for Immunology Inc. All rights reserved.


PubMed | Charles Darwin University, South Australia Health and Medical Research Institute, Flinders University, University of Adelaide and 2 more.
Type: Journal Article | Journal: International journal of pediatric otorhinolaryngology | Year: 2015

Indigenous Australian children have a high prevalence of otitis media with effusion (OME) and associated conductive hearing loss. Only three microbiological studies of middle ear fluid (MEF) from Indigenous Australian children with OME have been reported. All of these were reliant on culture or species-specific PCR assays. The aim of this study was to characterise the middle ear fluid (MEF), adenoid and nasopharyngeal (NP) microbiomes of Indigenous Australian children, using culture-independent 16S rRNA gene sequencing.MEF, NP swabs and adenoid specimens were collected from 11 children in the Alice Springs region of Central Australia. Bacterial communities in these specimens were characterised using 16S rRNA gene sequencing.The microbiota in MEF samples were dominated (>50% relative abundance) by operational taxonomic units (OTUs) consistent with Alloiococcus otitidis (6/11), Haemophilus influenzae (3/11) or Streptococcus sp. (specifically, Mitis group streptococci which includes Streptococcus pneumoniae) (1/11). Anatomical site selectivity was indicated by the presence of a single conserved Haemophilus OTU in 7/11 MEF samples. In comparison, there were ten distinct Haemophilus OTUs observed across the NP and adenoid samples. Despite significant differences between the MEF and NP/adenoid microbiomes, Streptococcus sp., H. influenzae and Moraxella catarrhalis OTUs were common to all sample types. Co-occurrence of classical otopathogens in paired MEF and NP/Adenoid samples is consistent with earlier culture-based studies.These data highlight the need to further assess H. influenzae traits important in otitis media and to understand the role of canal flora, especially A. otitidis, in populations with a high prevalence of tympanic membrane perforation.


Rogers G.B.,South Australia Health and Medical Research Institute | Rogers G.B.,Flinders University | Bruce K.D.,King's College London | Martin M.L.,Materials Adult Hospital | And 3 more authors.
The Lancet Respiratory Medicine | Year: 2014

Background: Long-term macrolide treatment has proven benefit in inflammatory airways diseases, but whether it leads to changes in the composition of respiratory microbiota is unknown. We aimed to assess whether long-term, low-dose erythromycin treatment changes the composition of respiratory microbiota in people with non-cystic fibrosis bronchiectasis. Methods: Microbiota composition was determined by 16S rRNA gene sequencing of sputum samples from participants in the BLESS trial, a 12-month, double-blind, placebo-controlled trial of twice-daily erythromycin ethylsuccinate (400 mg) in adult patients with non-cystic fibrosis bronchiectasis and at least two infective exacerbations in the preceding year. The primary outcome was within-patient change in respiratory microbiota composition (assessed by Bray-Curtis index) between baseline and week 48, comparing erythromycin with placebo. The BLESS trial is registered with the Australian New Zealand Clinical Trials Registry, number ACTRN12608000460303. Findings: The BLESS trial took place between Oct 15, 2008, and Dec 14, 2011. Paired sputum samples were available from 86 randomly assigned patients, 42 in the placebo group and 44 in the erythromycin group. The change in microbiota composition between baseline and week 48 was significantly greater with erythromycin than with placebo (median Bray-Curtis score 0·52 [IQR 0·14-0·78] vs 0·68 [0·46-0·93]; median difference 0·16, 95% CI 0·01-0·33; p=0·03). In patients with baseline airway infection dominated by Pseudomonas aeruginosa, erythromycin did not change microbiota composition significantly. In those with infection dominated by organisms other than P aeruginosa, erythromycin caused a significant change in microbiota composition (p=0·03 [by analysis of similarity]), representing a reduced relative abundance of Haemophilus influenzae (35·3% [5·5-91·6] vs 6·7% [0·8-74·8]; median difference 12·6%, 95% CI 0·4-28·3; p=0·04; interaction p=0·02) and an increased relative abundance of P aeruginosa (0·02% [0·00-0·33] vs 0·13% [0·01-39·58]; median difference 6·6%, 95% CI 0·1-37·1; p=0·002; interaction p=0·45). Compared with placebo, erythromycin reduced the rate of pulmonary exacerbations over the 48 weeks of the study in patients with P aeruginosa-dominated infection (median 1 [IQR 0-3] vs 3 [2-5]; median difference -2, 95% CI -4 to -1; p=0·01), but not in those without P aeruginosa-dominated infection (1 [0-2] vs 1 [0-3]; median difference 0, -1 to 0; p=0·41; interaction p=0·04). Interpretation: Long-term erythromycin treatment changes the composition of respiratory microbiota in patients with bronchiectasis. In patients without P aeruginosa airway infection, erythromycin did not significantly reduce exacerbations and promoted displacement of H influenzae by more macrolide-tolerant pathogens including P aeruginosa. These findings argue for a cautious approach to chronic macrolide use in patients without P aeruginosa airway infection. Funding: Mater Adult Respiratory Research Trust Fund. © 2014 Elsevier Ltd.


PubMed | Hannover Medical School, Institute Paoli Calmettes, Catholic University of Korea, South Australia Health and Medical Research Institute and 13 more.
Type: Journal Article | Journal: Haematologica | Year: 2016

To characterize the incidence, clinical features and genetics of ETV6-ABL1 leukemias, representing targetable kinase-activating lesions, we analyzed 44 new and published cases of ETV6-ABL1-positive hematologic malignancies [22 cases of acute lymphoblastic leukemia (13 children, 9 adults) and 22 myeloid malignancies (18 myeloproliferative neoplasms, 4 acute myeloid leukemias)]. The presence of the ETV6-ABL1 fusion was ascertained by cytogenetics, fluorescence in-situ hybridization, reverse transcriptase-polymerase chain reaction and RNA sequencing. Genomic and gene expression profiling was performed by single nucleotide polymorphism and expression arrays. Systematic screening of more than 4,500 cases revealed that in acute lymphoblastic leukemia ETV6-ABL1 is rare in childhood (0.17% cases) and slightly more common in adults (0.38%). There is no systematic screening of myeloproliferative neoplasms; however, the number of ETV6-ABL1-positive cases and the relative incidence of acute lymphoblastic leukemia and myeloproliferative neoplasms suggest that in adulthood ETV6-ABL1 is more common in BCR-ABL1-negative chronic myeloid leukemia-like myeloproliferations than in acute lymphoblastic leukemia. The genomic profile of ETV6-ABL1 acute lymphoblastic leukemia resembled that of BCR-ABL1 and BCR-ABL1-like cases with 80% of patients having concurrent CDKN2A/B and IKZF1 deletions. In the gene expression profiling all the ETV6-ABL1-positive samples clustered in close vicinity to BCR-ABL1 cases. All but one of the cases of ETV6-ABL1 acute lymphoblastic leukemia were classified as BCR-ABL1-like by a standardized assay. Over 60% of patients died, irrespectively of the disease or age subgroup examined. In conclusion, ETV6-ABL1 fusion occurs in both lymphoid and myeloid leukemias; the genomic profile and clinical behavior resemble BCR-ABL1-positive malignancies, including the unfavorable prognosis, particularly of acute leukemias. The poor outcome suggests that treatment with tyrosine kinase inhibitors should be considered for patients with this fusion.


PubMed | Baker Heart and Diabetes Institute, Monash University, Prince of Wales Hospital and South Australia Health and Medical Research Institute
Type: Journal Article | Journal: BMC health services research | Year: 2017

Valvular heart disease, including rheumatic heart disease (RHD), is an important cause of heart disease globally. Management of advanced disease can include surgery and other interventions to repair or replace affected valves. This article summarises the methodology of a study that will incorporate enhanced data collection systems to provide additional insights into treatment choice and outcome for advanced valvular disease including that due to RHD.An enhanced data collection system will be developed linking an existing Australian cardiac surgery registry to more detailed baseline co-morbidity, medication, echocardiographic and hospital separation data to identify predictors of morbidity and mortality outcome following valve surgery.This project aims to collect and incorporate more detailed information regarding pre and postoperative factors and subsequent morbidity. We will use this to provide additional insights into treatment choice and outcome.


PubMed | University of New South Wales, South Australia Health and Medical Research Institute, NCI Inc, St Jude Childrens Research Hospital and 2 more.
Type: Journal Article | Journal: Cancer research | Year: 2016

Ph-like acute lymphoblastic leukemia (ALL) is a genetically defined high-risk ALL subtype with a generally poor prognosis. In this study, we evaluated the efficacy of birinapant, a small-molecule mimetic of the apoptotic regulator SMAC, against a diverse set of ALL subtypes. Birinapant exhibited potent and selective cytotoxicity against B-cell precursor ALL (BCP-ALL) cells that were cultured ex vivo or in vivo as patient-derived tumor xenografts (PDX). Cytotoxicity was consistently most acute in Ph-like BCP-ALL. Unbiased gene expression analysis of BCP-ALL PDX specimens identified a 68-gene signature associated with birinapant sensitivity, including an enrichment for genes involved in inflammatory response, hematopoiesis, and cell death pathways. All Ph-like PDXs analyzed clustered within this 68-gene classifier. Mechanistically, birinapant sensitivity was associated with expression of TNF receptor TNFR1 and was abrogated by interfering with the TNF/TNFR1 interaction. In combination therapy, birinapant enhanced the in vivo efficacy of an induction-type regimen of vincristine, dexamethasone, and L-asparaginase against Ph-like ALL xenografts, offering a preclinical rationale to further evaluate this SMAC mimetic for BCP-ALL treatment. Cancer Res; 76(15); 4579-91. 2016 AACR.


Kowalczyk K.M.,University of Manchester | Petersen J.,University of Manchester | Petersen J.,Flinders University | Petersen J.,South Australia Health and Medical Research Institute
PLoS Genetics | Year: 2016

Target of Rapamycin (TOR) signalling allows eukaryotic cells to adjust cell growth in response to changes in their nutritional and environmental context. The two distinct TOR complexes (TORC1/2) localise to the cell’s internal membrane compartments; the endoplasmic reticulum (ER), Golgi apparatus and lysosomes/vacuoles. Here, we show that Ppk32, a SCYL family pseudo-kinase, is a novel regulator of TOR signalling. The absence of ppk32 expression confers resistance to TOR inhibition. Ppk32 inhibition of TORC1 is critical for cell survival following Brefeldin A (BFA) induced stress. Treatment of wild type cells with either the TORC1 specific inhibitor rapamycin or the general TOR inhibitor Torin1 confirmed that a reduction in TORC1 activity promoted recovery from BFA induced stress. Phosphorylation of Ppk32 on two residues that are conserved within the SCYL pseudo-kinase family are required for this TOR inhibition. Phosphorylation on these sites controls Ppk32 protein levels and sensitivity to BFA. BFA induced ER stress does not account for the response to BFA that we report here, however BFA is also known to induce Golgi stress and impair traffic to lysosomes. In summary, Ppk32 reduce TOR signalling in response to BFA induced stress to support cell survival. © 2016 Kowalczyk, Petersen.


Du W.,University of Manchester | Du W.,Qingdao University | Forte G.M.,University of Manchester | Smith D.,Cancer Research UK Research Institute | And 3 more authors.
Open Biology | Year: 2016

Cell proliferation, metabolism, migration and survival are coordinated through the tight control of two target of rapamycin (TOR) kinase complexes: TORC1 and TORC2. Here, we show that a novel phosphorylation of fission yeast Gad8 (AGC kinase) on the evolutionarily conserved threonine 6 (Thr6) prevents the physical association between Gad8 and TORC2. Accordingly, this block to protein interactions by Gad8 Thr6 phosphorylation decreases TORC2-controlled activation of Gad8. Likewise, phosphorylation of Gad8 Thr6, possibly by PKC, prevents the association of Gad8 with TORC2 thereby increasing TORC2 activity, because it reduces Gad8-mediated feedback inhibition of TORC2. Consistently, the introduction of a Gad8 T6D mutant, that mimics phosphorylation, increased TORC2 activity. Increased PKCPck2 expression prevented Gad8-TORC2 binding and so reduced the TORC2-mediated phosphorylation of Gad8 serine 546 that activates Gad8. Interestingly, independent of the Ser546 phosphorylation status, Gad8 Thr6 phosphorylation is important for remodelling the actin cytoskeleton and survival upon potassium ion and heat stresses. In contrast, Ser546 phosphorylation is required for the control of Gl arrest, mating, cell length at division and vascular size. Finally, these findings reveal a novel mode of TORC2 activation that is essential for cell survival following stress. © 2016 The Authors.

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