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1.  Structural mass spectrometry of tissue extracts to distinguish cancerous and non-cancerous breast diseases 
Molecular bioSystems  2014;10(11):2827-2837.
Breast cancer is well-known to broadly impact cellular metabolism and aberrant metabolism in breast cancer tumors has been widely studied by both targeted and untargeted analyses to characterize the affected metabolic pathways. In this work, we utilize ultra-performance liquid chromatography (UPLC) in tandem with ion mobility-mass spectrometry (IM-MS), which provides chromatographic, structural, and mass information, to characterize the aberrant metabolism associated with breast diseases such as cancer. In a double-blind analysis of matched control (n=3) and disease tissues (n=3), tissues were homogenized, polar metabolites were extracted, and the extracts were characterized by UPLC-IM-MS/MS. Principle component analysis revealed a strong separation between disease tissues, with one diseased tissue clustering with the control tissues along PC1 and two others separated along PC2. Using postion mobility MS/MS spectra acquired by data-independent acquisition, the features giving rise to the observed grouping were determined to be biomolecules associated with aggressive breast cancer tumors, including glutathione, oxidized glutathione, thymosins β4 and β10, and choline-containing species. Pathology reports revealed the outlier of the disease tissues to be a benign fibroadenoma, whereas the other disease tissues represented highly metabolic benign and aggressive tumors. This IM-MS-based workflow bridges the transition from untargeted metabolomic profiling to tentative identifications of key descriptive molecular features using data acquired in one analysis, with additional experiments performed only for validation. The ability to resolve cancerous and non-cancerous tissues at the biomolecular level demonstrates UPLC-IM-MS/MS as a robust and sensitive platform for metabolomic profiling of tissues.
PMCID: PMC4183709  PMID: 25212505
2.  How and When Do Insects Rely on Endogenous Protein and Lipid Resources during Lethal Bouts of Starvation? A New Application for 13C-Breath testing 
PLoS ONE  2015;10(10):e0140053.
Most of our understanding about the physiology of fasting and starvation comes from studies of vertebrates; however, for ethical reasons, studies that monitor vertebrates through the lethal endpoint are scant. Insects are convenient models to characterize the comparative strategies used to cope with starvation because they have diverse life histories and have evolved under the omnipresent challenge of food limitation. Moreover, we can study the physiology of starvation through its natural endpoint. In this study we raised populations of five species of insects (adult grasshoppers, crickets, cockroaches, and larval beetles and moths) on diets labeled with either 13C-palmitic acid or 13C-leucine to isotopically enrich the lipids or the proteins in their bodies, respectively. The insects were allowed to become postabsorptive and then starved. We periodically measured the δ13C of the exhaled breath to characterize how each species adjusted their reliance on endogenous lipids and proteins as energy sources. We found that starving insects employ a wide range of strategies for regulating lipid and protein oxidation. All of the insects except for the beetle larvae were capable of sharply reducing reliance on protein oxidation; however, this protein sparing strategy was usually unsustainable during the entire starvation period. All insects increased their reliance on lipid oxidation, but while some species (grasshoppers, cockroaches, and beetle larvae) were still relying extensively on lipids at the time of death, other species (crickets and moth larvae) allowed rates of lipid oxidation to return to prestarvation levels. Although lipids and proteins are critical metabolic fuels for both vertebrates and insects, insects apparently exhibit a much wider range of strategies for rationing these limited resources during starvation.
PMCID: PMC4605643  PMID: 26465334
3.  Restricted isotype, distinct variable gene usage, and high rate of gp120-specificity of HIV-1 Envelope-specific B cells in colostrum compared to those in blood of HIV-1-infected, lactating African women 
Mucosal immunology  2014;8(2):316-326.
A successful HIV-1 vaccine must elicit immune responses that impede mucosal virus transmission, though functional roles of protective HIV-1 Envelope (Env)-specific mucosal antibodies remain unclear. Colostrum is a rich source of readily accessible mucosal B cells that may help define the mucosal antibody response contributing to prevention of postnatal HIV-1 transmission. To examine the HIV-1 Env-specific colostrum B cell repertoire, single B cells were isolated from 17 chronically HIV-infected, lactating women, producing 51 blood and 39 colostrum HIV-1 Env-specific B cell antibodies. All HIV-1 Env-specific colostrum-derived antibodies were IgG1 isotype and had mean heavy chain complementarity-determining region 3 (CDR3) lengths and mutation frequencies similar to those isolated from blood. However, variable heavy chain (VH) gene subfamily 1~69 usage was higher among colostrum than blood HIV-1 Env-reactive antibodies (49% versus 20%, p = 0.006, Fisher’s exact test). Additionally, more HIV-1 Env-specific colostrum antibodies were gp120-specific than those isolated from blood (44% versus 16%, p = 0.005, Fisher’s exact test). One cross-compartment HIV-1 Env-specific clonal B cell lineage was identified. These unique characteristics of colostrum B cell antibodies suggest selective homing of HIV-1-specific IgG1-secreting memory B cells to the mammary gland and have implications for targeting mucosal B cell populations by vaccination.
PMCID: PMC4320043  PMID: 25100291
HIV-1; colostrum; B cell; antibodies
4.  Accumulation of Memory Precursor CD8 T Cells in Regressing Tumors Following Combination Therapy with Vaccine and Anti-PD-1 Antibody 
Cancer research  2014;74(11):2974-2985.
Immunosuppression in the tumor microenvironment blunts vaccine induced immune effectors. PD-1/B7-H1 is an important inhibitory axis in the tumor microenvironment. Our goal in this study was to determine the effect of blocking this inhibitory axis during and following vaccination against breast cancer. We observed that using anti-PD-1 antibody and a multi-peptide vaccine (consisting of immunogenic peptides derived from breast cancer antigens, neu, legumain and β-catenin) as a combination therapy regimen for the treatment of breast cancer bearing mice prolonged the vaccine-induced progression-free survival period. This prolonged survival was associated with increase in number of Tc1 and Tc2 CD8 T cells with memory precursor phenotype, CD27+IL-7RhiT-betlo and decrease in number of PD-1+ dendritic cells (DCs) in regressing tumors and enhanced antigen reactivity of tumor-infiltrating CD8 T cells. It was also observed that blockade of PD-1 on tumor DCs enhanced IL-7R expression on CD8 T cells. Taken together, our results suggest that PD-1 blockade enhances breast cancer vaccine efficacy by altering both CD8 T cell and DC components of the tumor microenvironment. Given the recent success of anti-PD-1 monotherapy, our results are encouraging for developing combination therapies for the treatment of cancer patients in which anti-PD-1 monotherapy alone may be ineffective (i.e. PD-L1-negative tumors).
PMCID: PMC4313351  PMID: 24728077
PD-1; peptides; vaccines; breast cancer; memory T cells
7.  Influence of Maternal Asthma and Asthma Severity on Newborn Morphometry 
To determine if maternal asthma or asthma severity affects newborn morphometry.
Study Design
A secondary analysis was performed on data collected in a multicenter prospective observational cohort study of asthma in pregnancy. Patients enrolled included women with asthma stratified by severity of disease and controls. Asthma severity was defined according to the classification proposed by the National Asthma Education Program (NAEP) Report of the Working Group on Asthma and Pregnancy, modified to include medication requirements. Newborn morphometry measurements included birth weight (BW) and multiples of the median birth weight (BW-MOM), head circumference (HC), length (L), HC:BW ratio, and ponderal index (PI).
Of 2480 patients there were 828 nonasthmatic controls, 828 with mild, 775 with moderate, and 49 with severe disease. Comparing all groups, there were statistically significant differences in maternal age (p < .001), race (p = .005), parity (p = .006), prepregnancy weight (p = .028), and medical care source (p = .001), with the severe asthma group having the highest mean maternal age (25.7 years), and proportion of African Americans (71.4%), proportion of multiparous patients (63.3%), and proportion of patients receiving government assistance (85.7%). When the control group was excluded from the comparisons, differences in prepregnancy weight and medical care source were no longer significant. BW-MOM and L did not differ between groups. The HC:BW ratio increased with asthma severity (p = .029) and was increased compared to controls (p = .010). This remained significant after controlling for confounding variables (both p <.001). HC was statistically significantly different between all groups (p = .032), as well as among women with varying degrees of asthma severity (p = .013), which was not clinically significant After covariates adjustment, HC was not significantly different among all groups (p = .228), nor the asthma groups (p = .144).
Asthma severity is associated with an increased HC:BW ratio. Severity was not found to impact HC, BW-MOM, L, or PI independently. However, the magnitudes of the effects were too small to suggest a clinically significant effect of asthma on neonatal morphometry in this large prospectively studied sample.
PMCID: PMC4349430  PMID: 20170320
asthma; neonatal morphometry; pregnancy
8.  An Approach to Evaluating and Comparing Biomarkers for Patient Treatment Selection 
Despite the heightened interest in developing biomarkers predicting treatment response that are used to optimize patient treatment decisions, there has been relatively little development of statistical methodology to evaluate these markers. There is currently no unified statistical framework for marker evaluation. This paper proposes a suite of descriptive and inferential methods designed to evaluate individual markers and to compare candidate markers. An R software package has been developed which implements these methods. Their utility is illustrated in the breast cancer treatment context, where candidate markers are evaluated for their ability to identify a subset of women who do not benefit from adjuvant chemotherapy and can therefore avoid its toxicity.
PMCID: PMC4341986  PMID: 24695044
9.  AXL induces epithelial to mesenchymal transition and regulates the function of breast cancer stem cells 
Oncogene  2013;33(10):1316-1324.
Despite significant progress in the treatment of breast cancer particularly through the use of targeted therapy, relapse and chemo-resistance remain a major hindrance to the fight to minimize the burden of the disease. It is becoming increasingly clear that a rare subpopulation of cells known as cancer stem cells (CSC), able to be generated through epithelial to mesenchymal transition (EMT) and capable of tumor initiation and self-renewal, contributes to treatment resistance and metastases. This means that a more effective therapy should target both the chemoresistant CSCs and the proliferating epithelial cells that give rise to them in order to reverse EMT and attenuate their conversion to CSCs. Here, we demonstrate a novel function of AXL in acting upstream to induce EMT in normal and immortalized human mammary epithelial cells in an apparent positive feedback loop mechanism and regulate breast CSC (BCSC) self-renewal and chemoresistance. Downregulation of AXL using MP470 (amuvatinib) reversed EMT in mesenchymal normal human mammary epithelial cells and murine BCSCs attenuating self-renewal and restored chemosensitivity of the BCSCs. AXL expression was also found to be associated with expression of stem cell genes, regulation of metastases genes, increased tumorigenicity, and was important for BCSC invasion and migration. Inactivation of AXL also led to downregulation of NFκB pathway and reduced tumor formation in vivo. Together, our data suggest that targeted therapy against AXL, in combination with systemic therapies, has the potential to improve response to anti-cancer therapies and to reduce breast cancer recurrence and metastases.
PMCID: PMC3994701  PMID: 23474758
AXL; EMT; CSCs; BCSCs; Tyrosine kinase
10.  Access to personalized medicine: factors influencing the use and value of gene expression profiling in breast cancer treatment 
Current Oncology  2014;21(3):e426-e433.
Genomic information is increasingly being used to personalize health care. One example is gene expression profiling (gep) tests, which estimate recurrence risk to inform chemotherapy decisions in breast cancer. Recently, gep tests were publicly funded in Ontario. We explored the perceived utility of gep tests, focusing on the factors influencing their use and value in treatment decision-making by patients and oncologists.
We conducted interviews with oncologists (n = 14) and interviews and a focus group with early-stage breast cancer patients (n = 28) who underwent gep testing. Both groups were recruited through oncology clinics in Ontario. Data were analyzed using the content analysis and constant comparison techniques.
Narratives from patients and oncologists provided insights into various factors facilitating and restricting access to gep. First, oncologists are positioned as gatekeepers of gep, providing access in medically appropriate cases. However, varying perceptions of appropriateness led to perceived inequities in access and negative impacts on the doctor–patient relationship. Second, media attention facilitated patient awareness of gep, but also complicated gatekeeping. Third, the dedicated administration attached to gep was burdensome and led to long waits for results and also to increased patient anxiety and delayed treatment. Collectively, because of barriers to access, those factors inadvertently heightened the perceived value of gep for patients relative to other prognostic indicators.
Our study delineates the factors facilitating and restricting access to gep, and highlights the roles of media and organization of services in the perceived value and utilization of gep. The results identify a need for administrative changes and practice guidelines to support streamlined and standardized use of gep tests.
PMCID: PMC4059806  PMID: 24940102
Gene expression profiling; breast cancer; patient perceptions; access; decision-making; genomics; risk recurrence; personalized medicine; health care providers; perspectives
11.  Patients’ perceptions of gene expression profiling in breast cancer treatment decisions 
Current Oncology  2014;21(2):e203-e211.
Determining the likely benefit of adjuvant chemotherapy for early-stage breast cancer patients depends on estimating baseline recurrence risk. Gene expression profile (gep) testing of tumours informs risk prediction, but evidence of its clinical utility is limited. We explored patient perceptions of gep testing and the impact of those perceptions on chemotherapy decisions.
We conducted one focus group (n = 4) and individual interviews (n = 24) with patients who used gep testing, recruited through clinics at two hospitals in Ontario. Data were analyzed using content analysis and constant comparison techniques.
Patients’ understanding of gep testing was variable, and misapprehensions were common. Patients valued the test because it provided them with certainty amidst confusion, with options and a sense of empowerment, and with personalized, authoritative information.
They commonly believed that the test was better and fundamentally different from other clinical tests, attributing to it unique power and truth-value. This kind of “magical thinking” was derived from an amplified perception of the test’s validity and patients’ need for reassurance about their treatment choices. Despite misperceptions or magical thinking, gep was widely considered to be the deciding factor in treatment decisions.
Patients tend to overestimate the truth-value of gep testing based on misperceptions of its validity. Our results identify a need to better support patient understanding of the test and its limitations. Findings illustrate the deep emotional investment patients make in gep test results and the impact of that investment on their treatment decisions.
PMCID: PMC3997453  PMID: 24764705
Gene expression profiling; breast cancer; patient perceptions; chemotherapy; decision-making; genomics; risk recurrence; personalized medicine
12.  Increased Protein-Coding Mutations in the Mitochondrial Genome of African American Women With Preeclampsia 
Reproductive Sciences  2012;19(12):1343-1351.
Preeclampsia occurs more frequently in women of African ancestry. The cause of this hypertensive complication is unclear, but placental oxidative stress may play a role. Because mitochondria are the major sites of oxidative phosphorylation, we hypothesized that placentas of preeclamptic pregnancies harbor mitochondrial DNA (mtDNA) mutations. Next-generation sequencing of placental mtDNA in African American preeclamptics (N = 30) and controls (N = 38) from Chicago revealed significant excesses in preeclamptics of nonsynonymous substitutions in protein-coding genes and mitochondrially encoded nicotinamide adenine dinucleotide dehydrogenase 5 gene and an increase in the substitution rate (P = .0001). Moreover, 88% of preeclamptics and 53% of controls carried at least one nonsynonymous substitution (P = .005; odds ratio [OR] = 6.36, 95% confidence interval [CI]: 1.5-39.1). These results were not replicated in a sample of African American preeclamptics (N = 162) and controls (N = 171) from Detroit. Differences in study design and heterogeneity may account for this lack of replication. Nonsynonymous substitutions in mtDNA may be risk factors for preeclampsia in some African American women, but additional studies are required to establish this relationship.
PMCID: PMC4046444  PMID: 22902742
preeclampsia; mtDNA; African American women; oxidative phosphorylation
13.  Utility of progranulin and serum leukocyte protease inhibitor as diagnostic and prognostic biomarkers in ovarian cancer 
Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer death in females and leading gynecologic cause of cancer death. Despite the identification of a number of serum biomarkers, methods to identify early stage disease and predict prognosis remain scarce. We have evaluated two biologically connected serum biomarkers, serum leukocyte protease inhibitor (SLPI) and progranulin (PGRN).
200 frozen plasma samples were acquired from the Mayo Clinic Biospecimen Repository for Ovarian Cancer Research. Samples were obtained from 50 patients with benign conditions, 50 with AJCC stage I and II EOC, and 100 with AJCC stage III and IV EOC patients. Samples were obtained prior to surgical resection of a mass and were analyzed for absolute levels of SLPI and PGRN using enzyme-linked immunosorbent (ELISA) assays. Receiver-operator characteristic curves were generated for SLPI and PGRN. Median follow-up was 48 months.
Absolute levels of SLPI were significantly elevated in patients with EOC compared to benign disease and predicted the presence of EOC (AUC of 0.812. P = 0.04); SLPI remained elevated in the subset of patients with normal CA-125, PGRN levels were not significantly increased in early stage or late stage EOC patients as a whole, but an increase in PGRN levels was associated with decreased overall survival in advanced EOC.
SLPI levels are elevated in epithelial ovarian cancer, and SLPI shows promise as a diagnostic biomarker for patients with both elevated and normal CA-125 levels. An increase in PGRN is associated with decreased overall survival.
SLPI is elevated in EOC and warrants investigation in a screening study in women at risk for EOC.
PMCID: PMC3839679  PMID: 23878295
Ovarian neoplasms; GRN protein, human; SLPI protein, human; Biological Markers; Prognosis
14.  What is the most dangerous snake? 
Letter to Editor of Journal of Venomous Animals and Toxins.
PMCID: PMC3766188  PMID: 23981479
Snakes; Snake bites; Snake venoms; Snake envenomation
15.  Unusually High Levels of n-6 Polyunsaturated Fatty Acids in Whale Sharks and Reef Manta Rays 
Lipids  2013;48(10):1029-1034.
Fatty acid (FA) signature analysis has been increasingly used to assess dietary preferences and trophodynamics in marine animals. We investigated FA signatures of connective tissue of the whale shark Rhincodon typus and muscle tissue of the reef manta ray Manta alfredi. We found high levels of n-6 polyunsaturated fatty acids (PUFA), dominated by arachidonic acid (20:4n-6; 12–17 % of total FA), and comparatively lower levels of the essential n-3 PUFA—eicosapentaenoic acid (20:5n-3; ~1 %) and docosahexaenoic acid (22:6n-3; 3–10 %). Whale sharks and reef manta rays are regularly observed feeding on surface aggregations of coastal crustacean zooplankton during the day, which generally have FA profiles dominated by n-3 PUFA. The high levels of n-6 PUFA in both giant elasmobranchs raise new questions about the origin of their main food source.
PMCID: PMC3779593  PMID: 23975574
n-3 Fatty acids; Arachidonic acid; Planktivores; Zooplankton; Elasmobranch
16.  A pitfall in the reconstruction of fibre ODFs using spherical deconvolution of diffusion MRI data 
Neuroimage  2013;65(C):433-448.
Diffusion weighted (DW) MRI facilitates non-invasive quantification of tissue microstructure and, in combination with appropriate signal processing, three-dimensional estimates of fibrous orientation. In recent years, attention has shifted from the diffusion tensor model, which assumes a unimodal Gaussian diffusion displacement profile to recover fibre orientation (with various well-documented limitations), towards more complex high angular resolution diffusion imaging (HARDI) analysis techniques.
Spherical deconvolution (SD) approaches assume that the fibre orientation density function (fODF) within a voxel can be obtained by deconvolving a ‘common’ single fibre response function from the observed set of DW signals. In practice, this common response function is not known a priori and thus an estimated fibre response must be used. Here the establishment of this single-fibre response function is referred to as ‘calibration’. This work examines the vulnerability of two different SD approaches to inappropriate response function calibration: (1) constrained spherical harmonic deconvolution (CSHD)—a technique that exploits spherical harmonic basis sets and (2) damped Richardson–Lucy (dRL) deconvolution—a technique based on the standard Richardson–Lucy deconvolution.
Through simulations, the impact of a discrepancy between the calibrated diffusion profiles and the observed (‘Target’) DW-signals in both single and crossing-fibre configurations was investigated. The results show that CSHD produces spurious fODF peaks (consistent with well known ringing artefacts) as the discrepancy between calibration and target response increases, while dRL demonstrates a lower over-all sensitivity to miscalibration (with a calibration response function for a highly anisotropic fibre being optimal). However, dRL demonstrates a reduced ability to resolve low anisotropy crossing-fibres compared to CSHD. It is concluded that the range and spatial-distribution of expected single-fibre anisotropies within an image must be carefully considered to ensure selection of the appropriate algorithm, parameters and calibration. Failure to choose the calibration response function carefully may severely impact the quality of any resultant tractography.
► We compare two spherical deconvolution based fODF retrieval techniques. ► We examine fODF error under inappropriate response function calibration. ► CSHD produces errors as response and target diffusion profiles diverge. ► dRL is poor against low FA targets but adequate across white matter. ► Manuscript updated to cover orientational dependence of CSHD errors.
PMCID: PMC3580290  PMID: 23085109
Spherical harmonic deconvolution; Richardson–Lucy; MRI; Calibration; Tractography; Diffusion tensor imaging
18.  Teaching Skeptical Inquiry 
CBE Life Sciences Education  2012;11(3):233-234.
PMCID: PMC3433294
19.  A randomised, phase II study of intetumumab, an anti-αv-integrin mAb, alone and with dacarbazine in stage IV melanoma 
British Journal of Cancer  2011;105(3):346-352.
αv integrins are involved in angiogenesis and melanoma tumourigenesis. Intetumumab (CNTO 95) is a fully human anti-αv-integrin monoclonal antibody.
In a multicentre, randomised, phase II study, stage IV melanoma patients were randomised 1 : 1 : 1 : 1 to 1000 mg m−2 dacarbazine+placebo (n=32), 1000 mg m−2 dacarbazine+10 mg kg−1 intetumumab (n=32), 10 mg kg−1 intetumumab (n=33), or 5 mg kg−1 intetumumab (n=32) q3w. The primary endpoint was progression-free survival (PFS). Secondary endpoints included overall survival (OS), objective response rate (ORR), adverse events, and pharmacokinetics.
No statistically significant differences in efficacy were observed between groups. In the dacarbazine+placebo, dacarbazine+intetumumab, 10 mg kg−1 intetumumab, and 5 mg kg−1 intetumumab groups, median PFS was 1.8, 2.5, 1.4, and 1.4 months; median OS was 8, 11, 15, and 9.8 months; and ORR of complete+partial response was 10, 3, 6, and 0%. Nonlinear intetumumab pharmacokinetics and potential intetumumab–dacarbazine interactions were observed. Transient, asymptomatic, nonrecurring, grade 1–2, uveitic reactions that resolved spontaneously or with topical steroids were seen in 22–30% of intetumumab-treated patients. Low-grade infusion-reaction symptoms (headache, fatigue, nausea, vomiting, fever, chills) were observed, as expected, in 16–73% of dacarbazine-treated patients. No intetumumab-related myelosuppression, laboratory/electrocardiogram abnormalities, or deaths occurred.
With its favourable safety profile and a nonsignificant trend towards improved OS, intetumumab merits further investigation in advanced melanoma.
PMCID: PMC3172894  PMID: 21750555
intetumumab; melanoma; αv integrins; dacarbazine; CNTO 95
20.  MHC class II epitope nesting modulates dendritic cell function and improves generation of antigen-specific CD4 helper T cells1 
CD4 T helper cells are critical to the development of coordinated immune responses to infections and tumors. T helper cells are activated through interactions of the TCR with MHC class II complexed with peptide. T cell activation is dependent on the density of MHC peptide complexes as well as the duration of interaction of the TCR with antigen presenting cells. In this study, we sought to determine whether MHC class II peptides could be modified with amino acid sequences that facilitated uptake and presentation with the goal of improving T helper cell activation in vitro and in vivo. A model epitope derived from the murine folate receptor alpha, a self and tumor antigen, was modified at its carboxy terminus with the invariant chain derived Ii-Key peptide and at its amino terminus with a peptide that enhances uptake of antigen by antigen presenting cells. Modification of peptide resulted in enhanced generation of high avidity murine folate receptor alpha T cells that persisted in vivo and homed to sites of antigen deposition. The nesting approach was epitope and species independent and specifically excluded expansion of CD4 regulatory T cells. The resulting T helper cells were therapeutic, enhanced in vivo helper activity, and had an increased ability to resist tolerizing immune microenvironments. In addition to improved immunoadjuvants, this epitope modification strategy may be useful for enhancing ex vivo and in vivo generation of T helper cells for preventing and treating diseases.
PMCID: PMC3119756  PMID: 21613617
21.  TGFβ/TNFα-Mediated Epithelial-Mesenchymal Transition Generates Breast Cancer Stem Cells with a Claudin-Low Phenotype 
Cancer research  2011;71(13):4707-4719.
Breast cancer recurrence is believed to be caused by a sub-population of cancer cells that possess the stem cell attribute of treatment resistance. Recently, we and others have reported the generation of breast cancer stem cells (BCSCs) by epithelial to mesenchymal transition (EMT), although the physiological process by which these cells may arise in vivo remains unclear. We show here that exposure of tumor cells to TGFβ and TNFα induces EMT and, more importantly, generates cells with a stable BCSC phenotype which is demonstrated by increased self-renewing capacity, greatly increased tumorigenicity, and increased resistance to oxaliplatin, etoposide and paclitaxel. Furthermore, gene expression analyses found that the TGFβ/TNFα-derived BCSCs showed down regulated expression of genes encoding Claudin 3, 4 and 7 and the luminal marker, cytokeratin 18. These changes indicate a shift to the claudin low molecular subtype, a recently identified breast cancer subtype characterized by the expression of mesenchymal and stem cell-associated markers and correlated with a poor prognosis. Taken together, the data show that cytokine exposure can be used to generate stable BCSCs ex vivo, and suggest that these cells may provide a valuable tool in the identification of stem cell-directed biomarkers and therapies in breast cancer.
PMCID: PMC3129359  PMID: 21555371
22.  Tumor infiltrating PD-1+ dendritic cells mediate immune suppression in ovarian cancer 
Within the ovarian cancer microenvironment there are several mechanisms that suppress the actions of anti-tumor immune effectors. Delineating the complex immune microenvironment is an important goal towards developing effective immune-based therapies. A dominant pathway of immune suppression in ovarian cancer involves tumor-associated and dendritic cell-associated, B7-H1. The interaction of B7-H1 with PD-1 on tumor-infiltrating T cells is a widely cited theory of immune suppression involving B7-H1 in ovarian cancer. Recent studies suggest that the B7-H1 ligand, PD-1, is also expressed on myeloid cells complicating interpretations of how B7-H1 regulates dendritic cell (DC) function in the tumor. In this study we found that ovarian cancer-infiltrating DCs progressively expressed increased levels of PD-1 over time in addition to B7-H1. These dual-positive PD-1+B7-H1+ DCs have a classical DC phenotype (i.e. CD11c+CD11b+CD8−) but are immature, suppressive and respond poorly to danger signals. Accumulation of PD-1+B7-H1+ DC in the tumor was associated with suppression of T cell activity and decreased infiltrating T cells in advancing tumors. T cell suppressor function of these DCs appeared to be mediated by T cell associated PD-1. In contrast, ligation of PD-1 expressed on the tumor-associated DC suppressed NFκB activation, release of immune regulatory cytokines, and upregulation of co-stimulatory molecules. PD-1 blockade in mice bearing ovarian cancer substantially reduced tumor burden and increased effector antigen-specific T cell responses. Our results reveal a novel role of tumor infiltrating PD-1+B7-H1+ DCs in mediating immune suppression in ovarian cancer.
PMCID: PMC3110549  PMID: 21551365
Tolerance; tumor microenvironment; dendritic cells
Hypertension in Pregnancy  2010;29(4):399-409.
Evaluate association of the inflammatory marker C-reactive protein with recurrent preeclampsia.
Serum samples collected longitudinally in women with previous preeclampsia from the Maternal-Fetal Medicine Units Network trial of aspirin to prevent preeclampsia were assayed for CRP.
Of 255 women studied, 50 developed recurrence. Baseline C-reactive protein concentration was similar between women who did and did not recur. After adjusting for confounders, neither elevated baseline C-reactive protein nor its change over gestation was associated with recurrence.
In this group of women with previous preeclampsia, neither baseline C-reactive protein concentration nor change in concentration over gestation was associated with recurrent preeclampsia.
PMCID: PMC3339658  PMID: 20701468
Pregnancy; Preeclampsia; C-Reactive Protein; Inflammation; Acute-Phase Reaction
24.  Mite dispersal among the Southern Ocean Islands and Antarctica before the last glacial maximum 
It has long been maintained that the majority of terrestrial Antarctic species are relatively recent, post last glacial maximum, arrivals with perhaps a few microbial or protozoan taxa being substantially older. Recent studies have questioned this ‘recolonization hypothesis’, though the range of taxa examined has been limited. Here, we present the first large-scale study for mites, one of two dominant terrestrial arthropod groups in the region. Specifically, we provide a broad-scale molecular phylogeny of a biologically significant group of ameronothroid mites from across the maritime and sub-Antarctic regions. Applying different dating approaches, we show that divergences among the ameronothroid mite genera Podacarus, Alaskozetes and Halozetes significantly predate the Pleistocene and provide evidence of independent dispersals across the Antarctic Polar Front. Our data add to a growing body of evidence demonstrating that many taxa have survived glaciation of the Antarctic continent and the sub-Antarctic islands. Moreover, they also provide evidence of a relatively uncommon trend of dispersals from islands to continental mainlands. Within the ameronothroid mites, two distinct clades with specific habitat preferences (marine intertidal versus terrestrial/supralittoral) exist, supporting a model of within-habitat speciation rather than colonization from marine refugia to terrestrial habitats. The present results provide additional impetus for a search for terrestrial refugia in an area previously thought to have lacked ice-free ground during glacial maxima.
PMCID: PMC3049079  PMID: 20943685
ameronothroid mites; biogeography; dispersal; glacial refugia; vicariance
25.  Casp8p41 expression in primary T cells induces a proinflammatory response 
AIDS (London, England)  2010;24(9):1251-1258.
HIV infection of CD4 T cells can lead to HIV protease-mediated cleavage of procaspase 8 generating a novel, HIV-specific peptide called Casp8p41. Casp8p41 has at least two biologic functions: induction of cell death via mitochondrial depolarization and release of cytochrome C, as well as activation of nuclear factor kappa B (NFκB). We have previously shown that Casp8p41-induced NFκB activation enhances HIV LTR transcription and consequently increases HIV replication. Herein, we questioned whether Casp8p41-induced NFκB activation impacts the cytokine profile of cells expressing Casp8p41.
Analysis of cells expressing Casp8p41 and HIV-infected T cells.
We assessed whether host genes are transcriptionally activated following Casp8p41 production, using microarray analysis, cytokine quantification, followed by western blot and flow cytometry.
Microarray analysis identified 259 genes significantly upregulated following expression of Casp8p41. Furthermore, Casp8p41 expression in primary CD4 T cells results in increased production of interleukin (IL)-2, IL-15 and tumor necrosis factor (TNF), as well as IL-1RA; whereas levels of granulocyte macrophage colony-stimulating factor and interferon (IFN)-γ were reduced in the Casp8p41 expressing cells. Intra-cellular flow cytometry confirmed the co-association of Casp8p41 with elevated TNF in HIV-infected cells.
These data indicate that the expression of Casp8p41 in HIV-infected CD4 T cells in addition to promoting apoptosis and enhancing HIV replication also promotes a proinflammatory cytokine milieu, which is characteristic of untreated HIV infection.
PMCID: PMC3150465  PMID: 20299954
apoptosis; Casp8p41; HIV; inflammation; protease; tumor necrosis factor

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