Real-time PCR complements immunohistochemistry in the determination of HER-2/neu status in breast cancer

BACKGROUND: The clinical benefit of determining the status of HER-2/neu amplification in breast cancer patients is well accepted. Although immunohistochemistry (IHC) is the most frequently used method to assess the over-expression of HER-2 protein, fluorescent in-situ hybridization (FISH) is recognized as the "gold standard" for the determining of HER-2/neu status. The greatest discordance between the two methods occurs among breast tumors that receive an indeterminate IHC score of 2+. More recently, a real-time polymerase chain reaction (PCR) assay using the LightCycler(® )has been developed for quantifying HER-2/neu gene amplification. In this study, we evaluated the sensitivity and specificity of a commercially available LightCycler assay as it compares to FISH. To determine whether this assay provides an accurate alternative for the determination of HER-2/neu status, we focused primarily on tumors that were deemed indeterminate or borderline status by IHC. METHODS: Thirty-nine breast tumors receiving an IHC score of 2+ were evaluated by both FISH and LightCycler(® )technologies in order to determine whether quantitative real-time PCR provides an accurate alternative for the determination of HER-2/neu status. RESULTS: We found a high concordance (92%) between FISH and real-time PCR results. We also observed that 10% of these tumors were positive for gene amplification by both FISH and real-time PCR. CONCLUSION: The data show that the results obtained for the gene amplification of HER-2/neu by real-time PCR on the LightCycler(® )instrument is comparable to results obtained by FISH. These results therefore suggest that real-time PCR analysis, using the LightCycler(®), is a viable alternative to FISH for reassessing breast tumors which receive an IHC score of 2+, and that a combined IHC and real-time PCR approach for the determination of HER-2 status in breast cancer patients may be an effective and efficient strategy

Identification of Intracellular and Plasma Membrane Calcium Channel Homologues in Pathogenic Parasites

Ca2+ channels regulate many crucial processes within cells and their abnormal activity can be damaging to cell survival, suggesting that they might represent attractive therapeutic targets in pathogenic organisms. Parasitic diseases such as malaria, leishmaniasis, trypanosomiasis and schistosomiasis are responsible for millions of deaths each year worldwide. The genomes of many pathogenic parasites have recently been sequenced, opening the way for rational design of targeted therapies. We analyzed genomes of pathogenic protozoan parasites as well as the genome of Schistosoma mansoni, and show the existence within them of genes encoding homologues of mammalian intracellular Ca2+ release channels: inositol 1,4,5-trisphosphate receptors (IP3Rs), ryanodine receptors (RyRs), two-pore Ca2+ channels (TPCs) and intracellular transient receptor potential (Trp) channels. The genomes of Trypanosoma, Leishmania and S. mansoni parasites encode IP3R/RyR and Trp channel homologues, and that of S. mansoni additionally encodes a TPC homologue. In contrast, apicomplexan parasites lack genes encoding IP3R/RyR homologues and possess only genes encoding TPC and Trp channel homologues (Toxoplasma gondii) or Trp channel homologues alone. The genomes of parasites also encode homologues of mammalian Ca2+ influx channels, including voltage-gated Ca2+ channels and plasma membrane Trp channels. The genome of S. mansoni also encodes Orai Ca2+ channel and STIM Ca2+ sensor homologues, suggesting that store-operated Ca2+ entry may occur in this parasite. Many anti-parasitic agents alter parasite Ca2+ homeostasis and some are known modulators of mammalian Ca2+ channels, suggesting that parasite Ca2+ channel homologues might be the targets of some current anti-parasitic drugs. Differences between human and parasite Ca2+ channels suggest that pathogen-specific targeting of these channels may be an attractive therapeutic prospect

Myocardial tagging by Cardiovascular Magnetic Resonance: evolution of techniques--pulse sequences, analysis algorithms, and applications

Cardiovascular magnetic resonance (CMR) tagging has been established as an essential technique for measuring regional myocardial function. It allows quantification of local intramyocardial motion measures, e.g. strain and strain rate. The invention of CMR tagging came in the late eighties, where the technique allowed for the first time for visualizing transmural myocardial movement without having to implant physical markers. This new idea opened the door for a series of developments and improvements that continue up to the present time. Different tagging techniques are currently available that are more extensive, improved, and sophisticated than they were twenty years ago. Each of these techniques has different versions for improved resolution, signal-to-noise ratio (SNR), scan time, anatomical coverage, three-dimensional capability, and image quality. The tagging techniques covered in this article can be broadly divided into two main categories: 1) Basic techniques, which include magnetization saturation, spatial modulation of magnetization (SPAMM), delay alternating with nutations for tailored excitation (DANTE), and complementary SPAMM (CSPAMM); and 2) Advanced techniques, which include harmonic phase (HARP), displacement encoding with stimulated echoes (DENSE), and strain encoding (SENC). Although most of these techniques were developed by separate groups and evolved from different backgrounds, they are in fact closely related to each other, and they can be interpreted from more than one perspective. Some of these techniques even followed parallel paths of developments, as illustrated in the article. As each technique has its own advantages, some efforts have been made to combine different techniques together for improved image quality or composite information acquisition. In this review, different developments in pulse sequences and related image processing techniques are described along with the necessities that led to their invention, which makes this article easy to read and the covered techniques easy to follow. Major studies that applied CMR tagging for studying myocardial mechanics are also summarized. Finally, the current article includes a plethora of ideas and techniques with over 300 references that motivate the reader to think about the future of CMR tagging

<it>Echinacea</it>-induced cytosolic Ca²⁺elevation in HEK293

<p>Abstract</p> <p>Background</p> <p>With a traditional medical use for treatment of various ailments, herbal preparations of <it>Echinacea </it>are now popularly used to improve immune responses. One likely mode of action is that alkamides from <it>Echinacea </it>bind to cannabinoid type 2 (CB2) receptors and induce a transient increase in intracellular Ca²⁺. Here, we show that unidentified compounds from <it>Echinacea purpurea </it>induce cytosolic Ca²⁺elevation in non-immune-related cells, which lack CB2 receptors and that the Ca²⁺elevation is not influenced by alkamides.</p> <p>Methods</p> <p>A non-immune human cell line, HEK293, was chosen to evaluate <it>E. purpurea </it>root extracts and constituents as potential regulators of intracellular Ca²⁺levels. Changes in cytosolic Ca²⁺levels were monitored and visualized by intracellular calcium imaging. U73122, a phospholipase C inhibitor, and 2-aminoethoxydiphenyl borate (2-APB), an antagonist of inositol-1,4,5-trisphosphate (IP₃) receptor, were tested to determine the mechanism of this Ca²⁺signaling pathway. <it>E. purpurea </it>root ethanol extracts were fractionated by preparative HPLC, screened for bioactivity on HEK293 cells and by GC-MS for potential constituent(s) responsible for this bioactivity.</p> <p>Results</p> <p>A rapid transient increase in cytosolic Ca²⁺levels occurs when <it>E. purpurea </it>extracts are applied to HEK293 cells. These stimulatory effects are phospholipase C and IP₃receptor dependent. <it>Echinacea</it>-evoked responses could not be blocked by SR 144528, a specific CB2 receptor antagonist, indicating that CB2 is not involved. Ca²⁺elevation is sustained after the <it>Echinacea</it>-induced Ca²⁺release from intracellular Ca²⁺stores; this longer-term effect is abolished by 2-APB, indicating a possible store operated calcium entry involvement. Of 28 HPLC fractions from <it>E. purpurea </it>root extracts, six induce cytosolic Ca²⁺increase. Interestingly, GC-MS analysis of these fractions, as well as treatment of HEK293 cells with known individual and combined chemicals, indicates the components thought to be responsible for the major immunomodulatory bioactivity of <it>Echinacea do not </it>explain the observed Ca²⁺response. Rather, lipophilic constituents of unknown structures are associated with this bioactivity.</p> <p>Conclusions</p> <p>Our data indicate that as yet unidentified constituents from <it>Echinacea </it>stimulate an IP₃receptor and phospholipase C mediation of cytosolic Ca²⁺levels in non-immune mammalian cells. This pathway is distinct from that induced in immune associated cells via the CB2 receptor.</p