Detection of breast cancer cells using targeted magnetic nanoparticles and ultra-sensitive magnetic field sensors.

INTRODUCTION: Breast cancer detection using mammography has improved clinical outcomes for many women, because mammography can detect very small (5 mm) tumors early in the course of the disease. However, mammography fails to detect 10 - 25% of tumors, and the results do not distinguish benign and malignant tumors. Reducing the false positive rate, even by a modest 10%, while improving the sensitivity, will lead to improved screening, and is a desirable and attainable goal. The emerging application of magnetic relaxometry, in particular using superconducting quantum interference device (SQUID) sensors, is fast and potentially more specific than mammography because it is designed to detect tumor-targeted iron oxide magnetic nanoparticles. Furthermore, magnetic relaxometry is theoretically more specific than MRI detection, because only target-bound nanoparticles are detected. Our group is developing antibody-conjugated magnetic nanoparticles targeted to breast cancer cells that can be detected using magnetic relaxometry. METHODS: To accomplish this, we identified a series of breast cancer cell lines expressing varying levels of the plasma membrane-expressed human epidermal growth factor-like receptor 2 (Her2) by flow cytometry. Anti-Her2 antibody was then conjugated to superparamagnetic iron oxide nanoparticles using the carbodiimide method. Labeled nanoparticles were incubated with breast cancer cell lines and visualized by confocal microscopy, Prussian blue histochemistry, and magnetic relaxometry. RESULTS: We demonstrated a time- and antigen concentration-dependent increase in the number of antibody-conjugated nanoparticles bound to cells. Next, anti Her2-conjugated nanoparticles injected into highly Her2-expressing tumor xenograft explants yielded a significantly higher SQUID relaxometry signal relative to unconjugated nanoparticles. Finally, labeled cells introduced into breast phantoms were measured by magnetic relaxometry, and as few as 1 million labeled cells were detected at a distance of 4.5 cm using our early prototype system. CONCLUSIONS: These results suggest that the antibody-conjugated magnetic nanoparticles are promising reagents to apply to in vivo breast tumor cell detection, and that SQUID-detected magnetic relaxometry is a viable, rapid, and highly sensitive method for in vitro nanoparticle development and eventual in vivo tumor detection.

Characterization of a new curtovirus, pepper yellow dwarf virus, from chile pepper and distribution in weed hosts in New Mexico.

Over 4,950 asymptomatic weed samples from more than 20 weed species that are host plants for curtoviruses were collected from ten chile pepper fields in southern New Mexico (NM) during 2003, 2004 and 2005 to identify whether they were infected with curtoviruses and to determine which curtoviruses were distributed in the weed population. Polymerase chain reaction using primers designed to detect a portion of the coat protein (cp) gene were used to detect curtoviruses, and infected plants were further tested for specific curtoviruses using primers designed to detect to a portion of the replication-associated protein (rep) gene. Amplification of the cp gene was successful from 3.7, 1.17, and 1.9% of the weed samples in 2003, 2004, and 2005, respectively. Seventy-three amplicons from those samples were sequenced and compared to well-characterized curtoviruses. Analysis of the rep nucleotide sequences showed that approximately 32.9% of the weed isolates tested were closely related to beet mild curly top virus (BMCTV). Approximately 12.4% were closely related to beet severe curly top virus (BSCTV). The rest of the weed isolates (54.7%), which shared a very high level of nucleotide sequence identity to each other, represent a new curtovirus species. Using eight primers designed for PCR, complete genomes of three curtoviruses isolated from chile pepper samples representing the three groups of curtoviruses in southern New Mexico were sequenced. Comparisons of whole sequences of the genomes revealed that the DG2SW171601 isolate (2,929 nucleotides) was nearly identical to BMCTV-W4 (approximately 98% nucleotide sequence identity). The LRME27601 isolate (2,927 nucleotides) was most closely related to BSCTV (approximately 92% nucleotide sequence identity). The LJN17601 isolate (2,959 nucleotides) shared only from 49.9 to 88.8% nucleotide sequence identity with other well-characterized curtoviruses. Based on the accepted cut-off of 89%, we propose that the LJN17601 isolate is a member of a new curtovirus species. Chile peppers infected with this virus in the field express chlorotic stunting symptoms, so we propose the name pepper yellow dwarf virus (PeYDV). This new curtovirus species may be the result of mutations in the genome and recombination between BMCTV-W4 and BSCTV.

Development of antibody-tagged nanoparticles for detection of transplant rejection using biomagnetic sensors.

Organ transplantation is a life-saving procedure and the preferred method of treatment for a growing number of disease states. The advent of new immunosuppressants and improved care has led to great advances in both patient and graft survival. However, acute T-cell-mediated graft rejection occurs in a significant quantity of recipients and remains a life-threatening condition. Acute rejection is associated with decrease in long-term graft survival, demonstrating a need to carefully monitor transplant patients. Current diagnostic criteria for transplant rejection rely on invasive tissue biopsies or relatively nonspecific clinical features. A noninvasive way is needed to detect, localize, and monitor transplant rejection. Capitalizing on advances in targeted contrast agents and magnetic-based detection technology, we developed anti-CD3 antibody-tagged nanoparticles. T cells were found to bind preferentially to antibody-tagged nanoparticles, as identified through light microscopy, transmission electron microscopy, and confocal microscopy. Using mouse skin graft models, we were also able to demonstrate in vivo vascular delivery of T-cell targeted nanoparticles. We conclude that targeting lymphocytes with magnetic nanoparticles is conducive to developing a novel, noninvasive strategy for identifying transplant rejection.

Two new species of Undifilum, fungal endophytes of Astragalus (locoweeds) in the United States.

New species of Undifilum, from locoweeds Astragalus lentiginosus Vitman and Astragalus mollissimus Torr., are described using morphological characteristics and molecular phylogenetic analyses as Undifilum fulvum Baucom & Creamer sp. nov. and Undifilum cinereum Baucom & Creamer sp. nov. Fungi were isolated from dried plants of A. lentiginosus var. araneosus, diphysus, lentiginosus, and wahweapensis collected from Arizona, Oregon, and Utah, USA, and A. mollissimus var. biglovii, earleii, and mollissimus collected from New Mexico, Oklahoma, and Texas, USA. Endophytic fungi from Astragalus locoweeds were compared to Undifilum oxytropis isolates obtained from dried plant material of Oxytropis lamberteii from New Mexico and Oxytropis sericea from Arizona, Colorado, New Mexico, Utah, and Wyoming. Extremely slow growth in vitro was observed for all, and conidia, if present, were ellipsoid with transverse septa. However, in vitro color, growth on four different media, and conidium size differed between fungi from Astragalus spp. and U. oxytropis. Neighbor-joining analyses of internal transcribed spacer (ITS) region and glyceraldehyde-3-phosphate dehydrogenase (GPD) gene sequences revealed that U. fulvum and U. cinereum formed a clade distinct from U. oxytropis. This was supported by neighbor-joining analyses of results generated from random amplified polymorphic DNA (RAPD) fragments using two different primers.