Detection of epithelial cancer cells in peripheral blood by reverse transcriptase-polymerase chain reaction.

Circulating cancer cells in the blood play a central role in the metastatic process. Their number can be very small and techniques for their detection need to be both sensitive and specific. Polymerase chain reaction (PCR) has been successfully used to detect small numbers of tumour cells in haematological cancer in which abnormalities in DNA are sufficiently consistent to make this possible. For most solid tumours this not yet feasible. However, we have found that reverse transcriptase (RT)-PRC for tissue-specific gene expression is a useful technique for identifying small numbers of circulating cells in melanoma and neuroblastoma patients. In this report we describe detection of colon carcinoma cells by RT-PCR using CK 20 mRNA as a marker. Unlike other cytokeratin genes examined (CK 8 and CK 19), CK 20 was not transcribed in normal haematopoietic cells. This suggests a role for RT-PCR in the detection of colon carcinoma metastasis in blood and bone marrow, using CK 20 as the target gene. Future analysis of clinical material will determine the clinical significance of this technique

High-latitude E Region Ionosphere-thermosphere Coupling: A Comparative Study Using in Situ and Incoherent Scatter Radar Observations

We present in situ and ground-based measurements of the ratio k of ion cyclotronangular frequency to ion-neutral momentum transfer collision frequency to investigateionosphere-thermosphere (IT) coupling in the auroral E region. In situ observations were obtained by NASA sounding rocket 36.234, which was launched into the nightsideE region ionosphere at 1229 UT on 19 January 2007 from Poker Flat, AK. The payload carried instrumentation to determine ion drift angle and electric field vectors. Neutral winds were measured by triangulating a chemical tracer released from rocket 41.064 launched two minutes later. k is calculated from the rotation of the ion drift angle relative to the E-cross-B drift direction in a frame co-rotating with the payload. Between the altitudes of 118 km and 130 km k increases exponentially with a scale height of 9.3 +/- 0.7 km, deviating from an exponential above 130 km. k = 1 at an altitude z(sub0) of 119.9 +/- 0.5 km. The ratio was also estimated from Poker Flat Incoherent Scatter Radar (PFISR) measurements using the rotation of ion velocity with altitude. Exponential fits to the PFISR measurements made during the flight of 41.064 yield z(sub0) 115.9 +/- 1.2 km and a scale height of 9.1 +/- 1.0 km. Differences between in situ and ground-based measurements show that the E region atmospheric densities were structured vertically and/or horizontally on scales of 1 km to 10 km. There were no signs of ionospheric structure in ion density or ion temperature below scales of 1 km. The observations demonstrate the accuracy with which the in situ and PFISR data may be used as probes of IT coupling

Consensus criteria for sensitive detection of minimal neuroblastoma cells in bone marrow, blood and stem cell preparations by immunocytology and QRT-PCR: recommendations by the International Neuroblastoma Risk Group Task Force

Disseminating disease is a predictive and prognostic indicator of poor outcome in children with neuroblastoma. Its accurate and sensitive assessment can facilitate optimal treatment decisions. The International Neuroblastoma Risk Group (INRG) Task Force has defined standardised methods for the determination of minimal disease (MD) by immunocytology (IC) and quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) using disialoganglioside GD2 and tyrosine hydroxylase mRNA respectively. The INRG standard operating procedures (SOPs) define methods for collecting, processing and evaluating bone marrow (BM), peripheral blood (PB) and peripheral blood stem cell harvest by IC and QRT-PCR. Sampling PB and BM is recommended at diagnosis, before and after myeloablative therapy and at the end of treatment. Peripheral blood stem cell products should be analysed at the time of harvest. Performing MD detection according to INRG SOPs will enable laboratories throughout the world to compare their results and thus facilitate quality-controlled multi-centre prospective trials to assess the clinical significance of MD and minimal residual disease in heterogeneous patient groups

Strong Magnetic Field Fluctuations within Filamentary Auroral Density Cavities Interpreted as VLF Saucer Sources

The Geoelectrodynamics and Electro-Optical Detection of Electron and SuprathermalIon Currents (GEODESIC) sounding rocket encountered more than 100 filamentary densitycavities associated with enhanced plasma waves at ELF (3 kHz) and VLF (310 kHz)frequencies and at altitudes of 800990 km during an auroral substorm. These cavities weresimilar in size (20 m diameter in most cases) to so-called lower-hybrid cavities (LHCs)observed by previous sounding rockets and satellites; however, in contrast, many of theGEODESIC cavities exhibited up to tenfold enhancements in magnetic wave powerthroughout the VLF band. GEODESIC also observed enhancements of ELF and VLFelectric fields both parallel and perpendicular to the geomagnetic field B0 within cavities,though the VLF E field increases were often not as large proportionally as seen in themagnetic fields. This behavior is opposite to that predicted by previously published theoriesof LHCs based on passive scattering of externally incident auroral hiss. We argue thatthe GEODESIC cavities are active wave generation sites capable of radiating VLF wavesinto the surrounding plasma and producing VLF saucers, with energy supplied by cold,upward flowing electron beams composing the auroral return current. This interpretation issupported by the observation that the most intense waves, both inside and outside cavities,occurred in regions where energetic electron precipitation was largely inhibited orabsent altogether. We suggest that the wave-enhanced cavities encountered by GEODESICwere qualitatively different from those observed by earlier spacecraft because of thefortuitous timing of the GEODESIC launch, which placed the payload at apogee within asubstorm-related return current during its most intense phase, lasting only a few minutes

Dynamics of social class contempt in contemporary British television comedy

This is the author's accepted manuscript. The final published article is available from the link below. Copyright @ 2010 Taylor & Francis.British television comedy has often ridiculed the complexities and characteristics of social class structures and identities. In recent years, poor white socially marginalised groups, now popularly referred to as “chavs”, have become a prevalent comedy target. One of the most popular and controversial television “comedy chavs” is Little Britain's fictional teenage single mother, Vicky Pollard. This article examines the representation of Vicky Pollard in light of contemporary widespread abuse of the white working class. Highlighting the polysemic and ambivalent nature of Vicky Pollard's representation, the article argues that whilst Little Britain's characterisation of Vicky Pollard largely contributes to contemporary widespread demonisation of the working class, there are moments within Little Britain when a more sympathetic tone towards the poor working class may be read, and where chav identities are used to ridicule the pretensions, superficiality, and falsity of middle-class identities. The article concludes that television comedy has been, and continues to be, a significant vehicle through which serious concerns, anxieties, and questions about social class and class identities are discursively constructed and contested

Continuous Activation of the CD122/STAT-5 Signaling Pathway during Selection of Antigen-Specific Regulatory T Cells in the Murine Thymus

Signaling events affecting thymic selection of un-manipulated polyclonal natural CD25+foxp3+ regulatory T cells (nTreg) have not been established ex vivo. Here, we report a higher frequency of phosphorylated STAT-5 (pSTAT-5) in nTreg cells in the adult murine thymus and to a lesser extent in the periphery, compared to other CD4+CD8− subsets. In the neonatal thymus, the numbers of pSTAT-5+ cells in CD25+foxp3− and nTreg cells increased in parallel, suggesting that pSTAT-5+CD25+foxp3− cells might represent the precursors of foxp3+ regulatory T cells. This “specific” pSTAT-5 expression detected in nTreg cells ex vivo was likely due to a very recent signal given by IL-2/IL-15 cytokines in vivo since (i) it disappeared rapidly if cells were left unstimulated in vitro and (ii) was also observed if total thymocytes were stimulated in vitro with saturating amounts of IL-2 and/or IL-15 but not IL-7. Interestingly, STAT-5 activation upon IL-2 stimulation correlated better with foxp3 and CD122 than with CD25 expression. Finally, we show that expression of an endogenous superantigen strongly affected the early Treg cell repertoire but not the proportion of pSTAT-5+ cells within this repertoire. Our results reveal that continuous activation of the CD122/STAT-5 signaling pathway characterize regulatory lineage differentiation in the murine thymus

Melanoma-inhibiting activity (MIA) mRNA is not exclusively transcribed in melanoma cells: low levels of MIA mRNA are present in various cell types and in peripheral blood

The detection of minimal amounts of melanoma cells by tyrosinase reverse transcription polymerase chain reaction (RT-PCR) is seriously hampered by false negative reports in blood of melanoma patients with disseminated melanoma. Therefore, additional assays which make use of multiple melanoma markers are needed. It has been shown that introduction of multiple markers increases the sensitivity of detection. Melanoma inhibitory activity (MIA) is one such melanoma-specific candidate gene. To test the specificity of MIA PCR, we performed 30 and 60 cycles of PCR with two different sets of MIA specific primers on 19 melanoma and 16 non-melanoma cell lines. MIA mRNA was detected in 16 out of 19 melanoma cell lines and in seven out of 16 non-melanoma cell lines after 30 cycles of PCR. However, MIA mRNA could be detected in all cell lines after 60 cycles of PCR. Also, in 14 out of 14 blood samples of melanoma patients, five out of six blood samples of non-melanoma patients and in seven out of seven blood samples of healthy volunteers, MIA mRNA was detected after 60 cycles of PCR, whereas no MIA PCR product could be detected in any of the blood samples after 30 cycles of PCR. We conclude that low levels of MIA transcripts are present in various normal and neoplastic cell types. Therefore, MIA is not a suitable marker gene to facilitate the detection of minimal amounts of melanoma cells in blood or in target organs of the metastatic process. © 1999 Cancer Research Campaig

R1507, an Anti-Insulin-Like Growth Factor-1 Receptor (IGF-1R) Antibody, and EWS/FLI-1 siRNA in Ewing's Sarcoma: Convergence at the IGF/IGFR/Akt Axis

A subset of patients with Ewing's sarcoma responds to anti-insulin-like growth factor-1 receptor (IGF-1R) antibodies. Mechanisms of sensitivity and resistance are unknown. We investigated whether an anti-IGF-1R antibody acts via a pathway that could also be suppressed by small interfering (si) RNA against the EWS/FLI-1 fusion protein, the hallmark of Ewing's sarcoma. The growth of two Ewing's sarcoma cell lines (TC-32 and TC-71) was inhibited by the fully human anti-IGF-1R antibody, R1507 (clonogenic and MTT assays). TC-32 and TC-71 cells express high levels of IGF-2, while RD-ES and A4573 Ewing's cell lines, which were less responsive to R1507 in our assays, express low or undetectable IGF-2, respectively. TC-71 cells also expressed high levels of IGF-1R, and R1507 decreased steady-state levels of this receptor by internalization/degradation, an effect which was associated with a decrease in p-IGF-1R, p-IRS-1, and p-Akt. EWS/FLI-1 siRNA also decreased p-Akt, due to its ability to increase IGF-BP3 levels and subsequently decrease IGF-1 and IGF-2 levels, thus inhibiting signaling through p-IGF-1R. This inhibition correlated with growth suppression and apoptosis. The attenuation of Akt activation was confirmed in TC-71 and HEK-293 (human embryonic kidney) cells by transfecting them with IGF-1R siRNA. We conclude that antibodies and siRNA to IGF-1R, as well as siRNA to EWS/FLI-1, act via intersecting IGF/IGF-1R signals that suppress a common point in this pathway, namely the phosphorylation of Akt

Exosomal microRNAs from Longitudinal Liquid Biopsies for the Prediction of Response to Induction Chemotherapy in High-Risk Neuroblastoma Patients: A Proof of Concept SIOPEN Study

Despite intensive treatment, 50% of children with high-risk neuroblastoma (HR-NB) succumb to their disease. Progression through current trials evaluating the efficacy of new treatments for children with HR disease usually depends on an inadequate response to induction chemotherapy, assessed using imaging modalities. In this study, we sought to identify circulating biomarkers that might be detected in a simple blood sample to predict patient response to induction chemotherapy. Since exosomes released by tumor cells can drive tumor growth and chemoresistance, we tested the hypothesis that exosomal microRNA (exo-miRNAs) in blood might predict response to induction chemotherapy. The exo-miRNAs expression profile in plasma samples collected from children treated in HR-NBL-1/SIOPEN before and after induction chemotherapy was compared to identify a three exo-miRs signature that could discriminate between poor and good responders. Exo-miRNAs expression also provided a chemoresistance index predicting the good or poor prognosis of HR-NB patients