Effect of delayed entry on performance of the BACT/ALERT FAN PLUS bottles in the BACT/ALERT VIRTUO blood culture system.

Delayed entry of patient blood culture samples into a microbial detection system is unavoidable at times, due to off-shift staffing or transporting samples to centralized laboratories. Pre-incubation time and temperature of blood culture bottles are the most critical factors impacting recovery and detection of microorganisms. A total of 1377 BACT/ALERT® (BTA) Fastidious Antimicrobial Neutralization (FAN® PLUS) bottles (FA PLUS, FN PLUS, and PF PLUS) were tested after delayed entry times of 24 and 36 h at 20-25 °C (room temperature, RT) prior to loading into the BACT/ALERT® VIRTUO® microbial detection system (VIRTUO). Clinically relevant organisms were inoculated into bottles with 5-84 colony forming units (CFU) per bottle, and human blood (0 to 10 mL), and then loaded into the VIRTUO. When bottles were loaded without delay, a mean time to detection (TTD) of 9.6 h was observed. For delayed bottles, the TTD reported by the VIRTUO was added to the 24-h and 36-h delay times and resulted in average time to results of 32.5 h and 42.5 h, respectively. The FAN PLUS bottles in conjunction with the VIRTUO produced acceptable results when delays up to 24 h at 20-25 °C occur in loading.

Phenotype-directed analysis of genotype in early-onset, familial breast cancers.

UNLABELLED: Considerable heterogeneity of morphology and disease outcome exists within breast cancers (BC), which likely reflects variable molecular pathogeneses within this broad clinical group. AIM: To evaluate the underlying genomic alterations associated with familial, early-onset BC (EOBC) phenotypes, in order to improve the management of this disease. METHODS: Using hierarchical clustering of morphological and immunophenotypical parameters, 116 EOBC were stratified into six groups. Conventional and array-based comparative genomic hybridisation was used to analyse the genomic alterations. RESULTS: Specific areas of genomic imbalance were associated with individual phenotypes. The largest phenotypical group was high grade, oestrogen receptor and HER-2 negative. This group contained the majority of BRCA1 germline mutation-associated tumours and commonly showed loss of chromosomal regions 5cent-5q13, 5q14-22 and 4q28-32. High mitotic rate, an important indicator of tumour cell proliferation and poor prognosis, was associated with gain of 19p, mapped within 7 Mb of the telomere. This region contains the candidate oncogene CDC34, the protein product of which is involved in ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor, p27Kip1. CONCLUSION: Phenotype-based analysis can be used to determine the genetic changes important in subtypes of BC. Further, the different morphological phenotypes could act as a cost-effective surrogate for genotypical stratification to facilitate optimal management of this disease.

Candidate tumor-suppressor genes on chromosome arm 8p in early-onset and high-grade breast cancers.

Loss of genetic material from chromosome arm 8p occurs commonly in breast carcinomas, suggesting that this region is the site of one or more tumor-suppressor genes (TSGs). Comparative genomic hybridization analysis showed that 8p loss is more common in breast cancers from pre-menopausal compared with post-menopausal patients, as well as in high-grade breast cancers, regardless of the menopausal status. Subsequent high-resolution gene expression profiling of genes mapped to chromosome arm 8p, on an extended cohort of clinical tumor samples, indicated a similar dichotomy of breast cancer clinicopathologic types. Some of these genes showed differential downregulation in early-onset and later-onset, high-grade cancers compared with lower-grade, later-onset cancers. Three such genes were analysed further by in situ technologies, performed on tissue microarrays representing breast tumor and normal tissue samples. PCM1, which encodes a centrosomal protein, and DUSP4/MKP-2, which encodes a MAP kinase phosphatase, both showed frequent gene and protein loss in carcinomas. In contrast, there was an excess of cases showing loss of expression in the absence of reduced gene copy number of SFRP1, which encodes a dominant-negative receptor for Wnt-family ligands. These candidate TSGs may constitute some of the molecular drivers of chromosome arm 8p loss in breast carcinogenesis.

Complex CGH alterations on chromosome arm 8p at candidate tumor suppressor gene loci in breast cancer cell lines.

Loss of genetic material from chromosome arm 8p occurs frequently in human breast carcinomas, consistent with this region of the genome harboring one or more tumor suppressor genes (TSGs). We used the complementary techniques of microsatellite-based LOH, high-density FISH, and conventional CGH on 6 breast cancer cell lines (MCF7, SKBR3, T47D, MDA MB453, BT549, and BT474) to investigate the molecular cytogenetic changes occurring on chromosome 8 during tumorigenesis, with particular emphasis on 6 potential TSGs on 8p. We identified multiple alterations of chromosome 8, including partial or complete deletion of 8p or 8q, duplication of 8q, and isochromosome 8q. The detailed FISH analysis showed several complex rearrangements of 8p with differing breakpoints of varying proximity to the genes of interest. High rates of LOH were observed at markers adjacent to or within PCM1, DUSP4/MKP2, NKX3A, and DLC1, supporting their status as candidate TSGs. Due to the complex ploidy status of these cell lines, relative loss of 8p material detected by CGH did not always correlate with microsatellite-based LOH results. These results extend our understanding of the mechanisms accompanying the dysregulation of candidate tumor suppressor loci on chromosome arm 8p, and identify appropriate cellular systems for further investigation of their biological properties.

Helper T cell differentiation and the problem of cellular inheritance.

The quality of the helper T cell response against antigen can determine the outcomes of infectious, inflammatory, and autoimmune diseases. Mature Th1 and Th2 cell subsets are thought to arise from a common naive progenitor. In these precursor cells, effector cytokine genes appear to exist in a restrictive structure, which is determined by methylation of cytosine bases and higher-order structure of chromatin. The restrictive gene structures appear to be plastic, giving way to more active structures in some daughter cells. Some genetic loci, which are active in naive cells, however, become silenced during terminal differentiation. Both the derepression of silent loci and the silencing of active loci appear to be linked to the process of DNA replication. Future investigation will be directed toward understanding the way in which patterns of gene expression are altered or transmitted during the cell division of helper T lymphocytes.

LCC15-MB cells are MDA-MB-435: a review of misidentified breast and prostate cell lines.

Current stocks of the LCC15-MB cell line, which we originally isolated from a human breast-bone metastasis, were found to be genetically matched to the MDA-MB-435 cell line from the Lombardi Cancer Center (MDA-MB-435-LCC) using comparative genomic hybridisation, DNA microsatellite analysis and chromosomal number. LCC15-MB stocks used for our previously published studies as well as the earliest available LCC15-MB cells also showed identity to MDA-MB-435-LCC cells. The original karyotype reported for LCC15-MB cells was considerably different to that of MDA-MB-435 cells, indicating that the original LCC15-MB cells were lost to contamination by MDA-MB-435-LCC cells. Chromosome number is the simplest test to distinguish original LCC15-MB cells (n approximately 75) from MDA-MB-435 (n approximately 52). Collectively, our results prove that LCC15-MB cells currently available are MDA-MB-435 cells and we suggest their re-designation as MDA-MB-435-LCC15 cells. We also review the known misclassification of breast and prostate cancer cell lines to date and have initiated a register maintained at http://www.svi.edu.au/cell_lines_registry.doc.

Chromosomal gains and losses in ocular melanoma detected by comparative genomic hybridization in an Australian population-based study.

To define the location of potential oncogenes and tumor suppressor genes in ocular melanoma we carried out comparative genomic hybridization (CGH) analysis on a population-based series of 25 formalin-fixed, paraffin-embedded primary tumors comprising 17 choroidal, 2 ciliary body, 4 iris, and 2 conjunctival melanomas. Twelve (48%) of the 25 melanomas showed no chromosomal changes and 13 (52%) had at least one chromosomal gain or loss. The mean number of CGH changes in all tumors was 3.3, with similar mean numbers of chromosomal gains (1.5) and losses (1.8). The highest number of chromosomal changes (i.e., nine) occurred in a conjunctival melanoma and included four changes not observed in tumors at any other ocular site (gains in 22q and 11p and losses in 6p and 17p). The most frequent gains in all primary ocular melanomas were on chromosome arm 8q (69%), 6p (31%) and 8p (23%) and the most frequent losses were on 6q (38%), 10q (23%), and 16q (23%). The most common pairing was gain in 8p and gain in 8q, implying a whole chromosome copy number increase; gains in 8p occurred only in conjunction with gains in 8q. The smallest regions of copy number alteration were mapped to gain of 8q21 and loss of 6q21, 10q21, and 16q22. Sublocalization of these chromosomal changes to single-band resolution should accelerate the identification of genes involved in the genesis of ocular melanoma.

Evaluation of automated and manual DNA purification methods for detecting Ricinus communis DNA during ricin investigations.

In April of 2013, letters addressed to the President of United States and other government officials were intercepted and found to be contaminated with ricin, heightening awareness about the need to evaluate laboratory methods for detecting ricin. This study evaluated commercial DNA purification methods for isolating Ricinus communis DNA as measured by real-time polymerase chain reaction (PCR). Four commercially available DNA purification methods (two automated, MagNA Pure compact and MagNA Pure LC, and two manual, MasterPure complete DNA and RNA purification kit and QIAamp DNA blood mini kit) were evaluated. We compared their ability to purify detectable levels of R. communis DNA from four different sample types, including crude preparations of ricin that could be used for biological crimes or acts of bioterrorism. Castor beans, spiked swabs, and spiked powders were included to simulate sample types typically tested during criminal and public health investigations. Real-time PCR analysis indicated that the QIAamp kit resulted in the greatest sensitivity for ricin preparations; the MasterPure kit performed best with spiked powders. The four methods detected equivalent levels by real-time PCR when castor beans and spiked swabs were used. All four methods yielded DNA free of PCR inhibitors as determined by the use of a PCR inhibition control assay. This study demonstrated that DNA purification methods differ in their ability to purify R. communis DNA; therefore, the purification method used for a given sample type can influence the sensitivity of real-time PCR assays for R. communis.

Transcriptional activators of helper T cell fate are required for establishment but not maintenance of signature cytokine expression.

The stability of helper T cell fates is not well understood. Using conditional introduction of dominant-negative factors, we now show that T-bet and GATA-3 are far more critical in establishment than maintenance of IFN-gamma and IL-4 activity during Th1 and Th2 maturation, respectively. We also show that a genetic interaction between T-bet and its target Hlx seems to be required for Th1 maturation, but that Hlx may also be dispensable for maintenance of a transcriptionally permissive ifng gene. In parallel to progressive activator independence in the permissive lineage, the ifng gene becomes more recalcitrant to switching as the forbidden lineage matures. T-bet plus Hlx can disrupt ifng silencing when introduced into developing Th2 cells, but they fail to perturb ifng silencing in mature Th2 cells. In contrast, a hypermorphic allele of T-bet can reverse silencing of the ifng gene in mature Th2 cells. These results suggest that signature gene activity of helper T cells is initially plastic but later becomes epigenetically fixed and offer an initial strategy for inducing mature cells to switch their fate.

Cutting edge: a critical role for gene silencing in preventing excessive type 1 immunity.

Immunity often depends on proper cell fate choice by helper T lymphocytes. A naive cell, with minimal expression of IFN-gamma and IL-4, must give rise to progeny expressing high levels of either one, but not both, of those cytokines to defend against protozoan and helminthic pathogens, respectively. In the present study, we show that inactivation of the Mbd2 gene, which links DNA methylation and repressed chromatin, results in enhanced resistance to the protozoan parasite Leishmania major but impaired immunity to the intestinal helminth Trichuris muris. Helper T cells from methyl CpG-binding domain protein-2-deficient mice exhibit exuberant patterns of cytokine expression despite appropriate silencing of genes encoding the lineage-specifying factors T-bet and GATA-3. These results suggest that gene silencing can facilitate the ability of a progenitor cell to give rise to appropriately differentiated daughter cells in vivo. These findings also point to novel pathways that could participate in genetic control of resistance to infection and autoimmunity.

Gene silencing quantitatively controls the function of a developmental trans-activator.

How a single cell gives rise to progeny with differing fates remains poorly understood. We examined cells lacking methyl CpG binding domain protein-2 (MBD2), a molecule that has been proposed to link DNA methylation to silent chromatin. Helper T cells from Mbd2(-/-) mice exhibit disordered differentiation. IL-4, the signature of a restricted set of progeny, is expressed ectopically in Mbd2(-/-) parent and daughter cells. Loss of MBD2-mediated silencing renders the normally essential activator, Gata-3, dispensable for IL-4 induction. Gata-3 and MBD2 act in competition, wherein each factor independently, and quantitatively, regulates the binary choice of whether heritable IL-4 expression is established. Gata-3 functions, in part, to displace MBD2 from methylated DNA. These results suggest that activating and silencing signals integrate to provide spatially and temporally restricted patterns of gene activity.

Hlx is induced by and genetically interacts with T-bet to promote heritable T(H)1 gene induction.

Type 1 helper T (T(H)1) cells are essential for cellular immunity, but their ontogeny, maturation and durability remain poorly understood. By constructing a dominant-negative form of T-bet, we were able to determine the role played by this lineage-inducing trans-activator in the establishment and maintenance of heritable T(H)1 gene expression. Optimal induction of interferon-gamma (IFN-gamma) expression required genetic interaction between T-bet and its target, the homeoprotein Hlx. In fully mature T(H)1 cells, reiteration of IFN-gamma expression and stable chromatin remodeling became relatively independent of T-bet activity and coincided with demethylation of DNA. In contrast, some lineage attributes, such as expression of IL-12R beta 2 (interleukin 12 receptor beta 2), required ongoing T-bet activity in mature T(H)1 cells and their progeny. These findings suggest that heritable states of gene expression might be maintained by continued expression of the inducing factor or by a mechanism that confers a stable imprint of the induced state.

Detailed gene copy number and RNA expression analysis of the 17q12-23 region in primary breast cancers.

Chromosome region 17q12-23 commonly shows an increase in DNA copy number in breast cancers, suggesting that several oncogenes are located at this site. We performed a high-resolution expression array and comparative genomic hybridization analysis of genes mapped to the entire 17q12-23 region, to identify novel candidate oncogenes. We identified 24 genes that showed significant overexpression in breast cancers with gain of 17q12-23, compared to cancers without gain. These genes included previously identified oncogenes, together with several novel candidate oncogenes. FISH analysis using specific gene probes hybridized to tissue arrays confirmed the underlying amplification of overexpressed genes. This high-resolution analysis of the 17q12-23 region indicates that several established and novel candidate oncogenes, including a Wnt-signaling pathway member, are amplified and overexpressed within individual primary breast cancer samples. We were also able to confirm the presence of two apparently separate and reciprocally amplified groups of genes within this region. Investigation of these genes and their functional interactions will facilitate our understanding of breast oncogenesis and optimal management of this disease.

Control of effector CD8+ T cell function by the transcription factor Eomesodermin.

Activated CD8+ T cells play a critical role in host defense against viruses, intracellular microbes, and tumors. It is not clear if a key regulatory transcription factor unites the effector functions of CD8+ T cells. We now show that Eomesodermin (Eomes), a paralogue of T-bet, is induced in effector CD8+ T cells in vitro and in vivo. Ectopic expression of Eomes was sufficient to invoke attributes of effector CD8+ T cells, including interferon-gamma (IFN-gamma), perforin, and granzyme B. Loss-of-function analysis suggests Eomes may also be necessary for full effector differentiation of CD8+ T cells. We suggest that Eomesodermin is likely to complement the actions of T-bet and act as a key regulatory gene in the development of cell-mediated immunity.