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Smart C.E.,University of Queensland | Smart C.E.,The Queensland Institute of Medical Research QIMR | Morrison B.J.,The Queensland Institute of Medical Research QIMR | Morrison B.J.,Griffith University | And 17 more authors.
PLoS ONE | Year: 2013

Mammosphere and breast tumoursphere culture have gained popularity as in vitro assays for propagating and analysing normal and cancer stem cells. Whether the spheres derived from different sources or parent cultures themselves are indeed single entities enriched in stem/progenitor cells compared to other culture formats has not been fully determined. We surveyed sphere-forming capacity across 26 breast cell lines, immunophenotyped spheres from six luminal- and basal-like lines by immunohistochemistry and flow cytometry and compared clonogenicity between sphere, adherent and matrigel culture formats using in vitro functional assays. Analyses revealed morphological and molecular intra- and inter-sphere heterogeneity, consistent with adherent parental cell line phenotypes. Flow cytometry showed sphere culture does not universally enrich for markers previously associated with stem cell phenotypes, although we found some cell-line specific changes between sphere and adherent formats. Sphere-forming efficiency was significantly lower than adherent or matrigel clonogenicity and constant over serial passage. Surprisingly, self-renewal capacity of sphere-derived cells was similar/lower than other culture formats. We observed significant correlation between long-term-proliferating-cell symmetric division rates in sphere and adherent cultures, suggesting functional overlap between the compartments sustaining them. Experiments with normal primary human mammary epithelia, including sorted luminal (MUC1+) and basal/myoepithelial (CD10+) cells revealed distinct luminal-like, basal-like and mesenchymal entities amongst primary mammospheres. Morphological and colony-forming-cell assay data suggested mammosphere culture may enrich for a luminal progenitor phenotype, or induce reversion/relaxation of the basal/mesenchymal in vitro selection occurring with adherent culture. Overall, cell line tumourspheres and primary mammospheres are not homogenous entities enriched for stem cells, suggesting a more cautious approach to interpreting data from these assays and careful consideration of its limitations. Sphere culture may represent an alternative 3-dimensional culture system which rather than universally 'enriching' for stem cells, has utility as one of a suite of functional assays that provide a read-out of progenitor activity. © 2013 Smart et al. Source


Zaman M.,University of Queensland | Skwarczynski M.,University of Queensland | Malcolm J.M.,The Queensland Institute of Medical Research QIMR | Urbani C.N.,University of Queensland | And 5 more authors.
Nanomedicine: Nanotechnology, Biology, and Medicine | Year: 2011

Infection with Streptococcus pyogenes, commonly known as group A Streptococcus (GAS), is responsible for acute and postinfectious complications, including rheumatic fever and rheumatic heart disease (RHD). RHD is a global health burden, and Australia's indigenous population has one of the highest incidences of RHD worldwide. A potential peptide (J14) vaccine candidate has been previously identified from the C-terminal region of the M protein. However, such peptide-based vaccine development is hampered by a lack of carriers and adjuvants suitable for humans use. We have developed a fully synthetic peptide subunit vaccine candidate based on polyacrylate dendritic polymer. Intranasal administration of this nanoparticulate construct without additional adjuvant induced J14-specific IgG, which was also capable of in vitro opsonization of GAS, highlighting the potential of self-adjuvanting polyacrylate nanoparticle-based construct as a peptide vaccine delivery platform that may afford promising opportunities for treating systemic GAS infection. From the Clinical Editor: Polyacrylate dendrimers offer a unique approach to a nasally administered vaccine for addressing rheumatic heart disease. This paper describes the delivery of the J14 peptide, a C-terminal derivative of M-protein in group A Streptococcus. © 2011 Elsevier Inc. Source


Zaman M.,University of Queensland | Abdel-Aal A.-B.M.,University of Queensland | Phillipps K.S.M.,University of Queensland | Fujita Y.,University of Queensland | And 2 more authors.
Vaccine | Year: 2010

Incorporation of lipoamino acids (LAAs) into peptide structures effectively imparts self-adjuvanting activity onto otherwise ineffective immunogens. Our fully synthetic lipopeptide vaccine candidates against group A streptococcus (GAS) were composed of J14 as a target GAS B-cell epitope alongside a universal helper T-cell epitope (P25) and a LAA-based lipid moiety. In the current study, we investigated the ability of our lipopeptides to activate nuclear factor-κB (NF-κB) in a toll-like receptor-2 (TLR2)-dependent manner as the possible mode of action and reported the structure-function requirements for novel TLR2 targeting lipopeptides based on LAAs. The NF-κB activation was dependent on the dose and the length of the alkyl chains of the incorporated lipid moieties with the hierarchy LAA 3 (16 carbons) > LAA 2 (14 carbons) > LAA 1 (12 carbons). The position of the lipid moiety (C-terminus vs. Nε-terminus of the central lysine residue) does not significantly affect NF-κB activation. Lipopeptides containing different copies of LAA 3 were synthesized and the di-lipidated analogue was the most effective in NFκB activation. © 2010 Elsevier Ltd. All rights reserved. Source

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