Differential effects of HIF-α isoforms on apoptosis in renal carcinoma cell lines.

BACKGROUND: Germline mutations in the von Hippel-Lindau (VHL) tumor suppressor gene predispose individuals to clear cell renal carcinomas, hemangioblastomas, and pheochromocytomas. The VHL gene product forms an ubiquitin E3 ligase complex, with regulation of hypoxia-inducible factor alpha (HIF-α) as its best known function. Lack of VHL expression has been shown previously to sensitize renal cells to apoptosis caused by certain cellular stresses. In this report, the role of HIF-α in apoptosis was investigated using two parent VHL-null renal carcinoma cell lines. METHODS: 786-O and RCC10 renal carcinoma cell lines with manipulated levels of VHL, HIF-1α, or HIF-2α were subjected to cellular stresses and analyzed by western blotting for the abundance of apoptotic markers. RESULTS: Cell lines expressing mutant VHL proteins that were unable to regulate HIF-α had increased levels of apoptosis when irradiated with ultraviolet (UV) light. The influences of the two major isoforms of HIF-α, HIF-1α and HIF-2α, on apoptosis, were compared by creating cell lines in which levels of each isoform were modulated via short hairpin RNA interference. In UV-irradiated cells, HIF-2α expression was determined to promote apoptosis, whereas HIF-1α was anti-apoptotic. In cells deprived of either glucose or serum, HIF-1α expression was generally anti-apoptotic, while HIF-2α expression was observed to either promote apoptosis or have less of an influence on apoptosis, depending on the cell line used. CONCLUSIONS: HIF-1α and HIF-2α exerted distinct effects in each of the conditions tested, with expression of HIF-1α largely blocking apoptosis and HIF-2α generally promoting apoptosis. These results reinforce that HIF-1α and HIF-2α have distinct biological roles and that their relative expression levels may influence some therapeutic interventions that are dependent on apoptosis.

Toward product-based regulation of crops.

Current process-based approaches to regulation are no longer fit for purpose.

Catechol metabolites of endogenous estrogens induce redox cycling and generate reactive oxygen species in breast epithelial cells.

Estrogens are major risk factors for the development of breast cancer; they can be metabolized to catechols, which are further oxidized to DNA-reactive quinones and semiquinones (SQs). These metabolites are mutagenic and may contribute to the carcinogenic activity of estrogens. Redox cycling of the SQs and subsequent generation of reactive oxygen species (ROS) is also an important mechanism leading to DNA damage. The SQs of exogenous estrogens have been shown to redox cycle, however, redox cycling and the generation of ROS by endogenous estrogens has never been characterized. In the present studies, we determined whether the catechol metabolites of endogenous estrogens, including 2-hydroxyestradiol, 4-hydroxyestradiol, 4-hydroxyestrone and 2-hydroxyestriol, can redox cycle in breast epithelial cells. These catechol estrogens, but not estradiol, estrone, estriol or 2-methoxyestradiol, were found to redox cycle and generate hydrogen peroxide (H(2)O(2)) and hydroxyl radicals in lysates of three different breast epithelial cell lines: MCF-7, MDA-MB-231 and MCF-10A. The generation of ROS required reduced nicotinamide adenine dinucleotide phosphate as a reducing equivalent and was inhibited by diphenyleneiodonium, a flavoenzyme inhibitor, indicating that redox cycling is mediated by flavin-containing oxidoreductases. Using extracellular microsensors, catechol estrogen metabolites stimulated the release of H(2)O(2) by adherent cells, indicating that redox cycling occurs in viable intact cells. Taken together, these data demonstrate that catechol metabolites of endogenous estrogens undergo redox cycling in breast epithelial cells, resulting in ROS production. Depending on the localized concentrations of catechol estrogens and enzymes that mediate redox cycling, this may be an important mechanism contributing to the development of breast cancer.

A Pilot Study Testing a Novel 3D Printed Amphibious Lower Limb Prosthesis in a Recreational Pool Setting.

INTRODUCTION: Adults with limb amputation and other physical disabilities are less likely to participate in physical activity than adults in the general population and have elevated risk of heart disease and stroke. Swimming is a physical activity often recommended for persons with limb amputation. However, a standard economical swim prosthesis that facilitates easy transition from land to water does not exist. OBJECTIVE: The objectives were (1) to measure ease of first-time use and likability of a novel U.S. Food and Drug Administration (FDA)-cleared 510(k) three-dimensional (3D) printed device, the "FIN," in a recreational pool; and (2) to determine differences in time to complete basic swim tasks using the novel 3D printed amphibious lower limb prosthesis or a standard Swim Ankle prosthesis. Our hypotheses were the following: (1) that the novel 3D printed amphibious lower limb prosthesis would be easy and likeable upon first use; and (2) that basic swim tasks would take comparable time to complete with either device. SETTING: Academic medical center and community pool in New York. PARTICIPANTS: Participants were (N = 10) English-speaking adults with a transtibial amputation who self-identified to swim comfortably in a recreational setting. INTERVENTIONS: Participants completed tasks typical of recreational swimming while wearing the novel 3D printed amphibious lower limb prosthesis or a Swim Ankle. MAIN OUTCOME MEASUREMENTS: Participants performed a series of recreational swim tasks at self-selected speeds: entering/exiting pool, walking, swimming, and treading water, and completed a survey to assess the primary outcomes: likability, ease of use, and adverse events (feasibility). RESULTS: Participants found the novel 3D printed amphibious lower limb prosthesis more likable compared to the Swim Ankle and easy to use. Time to exit the pool was significantly reduced with the novel 3D printed amphibious lower limb prosthesis, while time to complete a 25-m lap was comparable. Participants did not show significant changes in vital signs when using either prosthesis. CONCLUSIONS: The novel 3D printed amphibious lower limb prosthesis was likable and easy to use upon first use. This study supports conducting a larger clinical trial to determine if the data are broadly reproducible.

Expert panel report on a study of Splenda in male rats.

A recent study in rats investigated the retail sweetener product, Granulated SPLENDA No Calorie Sweetener (Splenda) (Abou-Donia et al., 2008. Splenda alters gut microflora and increases intestinal P-glycoprotein and cytochrome P-450 in male rats. J. Toxicol. Environ. Health A, 71, 1415-1429), which is composed of (by dry weight) maltodextrin ( approximately 99%) and sucralose ( approximately 1%). The investigators reported that Splenda increased body weight, decreased beneficial intestinal bacteria, and increased the expression of certain cytochrome P450 (CYP450) enzymes and the transporter protein, P-glycoprotein (P-gp), the latter of which was considered evidence that Splenda or sucralose might interfere with the absorption of nutrients and drugs. The investigators indicated that the reported changes were attributable to the sucralose present in the product tested. An Expert Panel conducted a rigorous evaluation of this study. In arriving at its conclusions, the Expert Panel considered the design and conduct of the study, its outcomes and the outcomes reported in other data available publicly. The Expert Panel found that the study was deficient in several critical areas and that its results cannot be interpreted as evidence that either Splenda, or sucralose, produced adverse effects in male rats, including effects on gastrointestinal microflora, body weight, CYP450 and P-gp activity, and nutrient and drug absorption. The study conclusions are not consistent with published literature and not supported by the data presented.

Single variable domains from the T cell receptor ß chain function as mono- and bifunctional CARs and TCRs.

Cell therapy using T cell receptors (TCRs) and chimeric antigen receptors (CARs) represents a new wave of immunotherapies garnering considerable attention and investment. Further progress in this area of medicine depends in part on improving the functional capabilities of the engineered components, while maintaining the overall size of recombinant constructs to ensure their compatibility with existing gene delivery vehicles. We describe a single-variable-domain TCR (svd TCR) that utilizes only the variable domain of the ß chain (Vß). This Vß module not only works in TCR and CAR formats, but also can be used to create single-chain bispecific CARs and TCRs. Comparison of individual ligand-binding Vß domains in different formats suggests that the lone Vß sequence controls the sensitivity and a major part of the specificity of the CAR or TCR construct, regardless of signaling format, in Jurkat and primary T cells.

Clinical and biological activity of soy protein powder supplementation in healthy male volunteers.

PURPOSE: To determine if a commonly used soy protein supplement exhibits biological activity in vivo and in vitro, we evaluated an over-the-counter soy protein powder supplement using blood from healthy male volunteers and in an estrogen receptor in vitro assay. SUBJECTS AND METHODS: We recruited healthy male volunteers 18 years of age or older that were in good health. Treatment consisted of consuming two scoops (56 g) of pure soy protein powder (Puritan's Pride, Oakdale, NY) daily for 28 days. Serum testosterone and luteinizing hormone (LH) levels were collected on days -7, 0, 14, and 28 of therapy, and day 42. A reporter estrogen receptor (ER) assay was used to determine the effect on ER-beta and ER-alpha in vitro. RESULTS: Twelve subjects were enrolled with a mean age of 32.25 years (range 25 to 47). Serum testosterone decreased 19%(+/-22%) during the 4-week use of soy protein powder (P = 0.021) and increased within 2 weeks after we discontinued soy protein powder. Serum LH concentrations decreased during the 4-week use of soy protein powder then increased within 2 weeks after we stopped the soy protein powder, but the changes did not reach statistical significance (P = 0.20). Soy protein powder was found to induce agonist activity to ER-beta using a reporter estrogen receptor assay in yeast. CONCLUSION: Soy protein powder decreases serum testosterone levels in healthy men and acts as an ER-beta agonist; the significance of this biological effect with respect to cancer prevention needs further study.

Synergistic apoptosis of MCF-7 breast cancer cells by 2-methoxyestradiol and bis(ethyl)norspermine.

2-Methoxyestradiol (2ME) is an estradiol metabolite with anti-tumor and anti-angiogenic properties. We studied the effect of 2ME on apoptosis of MCF-7 breast cancer cells and explored a combination therapy using 2ME and a polyamine analogue, bis(ethyl)norspermine (BE-3-3-3). Determination of viable cells on day 4 of treatment with 2ME/BE-3-3-3 combinations showed synergistic effects by Chou-Talalay analysis. APO-BRDU analysis showed that there was only 1.5+/-0.5% apoptosis at 200 nM 2ME and 3.7+/-1.7% in the presence of 2.5 microM BE-3-3-3. Combination of 200 nM 2ME and 2.5 microM BE-3-3-3 resulted in 52.2+/-2.6% apoptosis. Up to 90% of the cells underwent apoptosis in the presence of 1000 nM 2ME and 2.5 microM BE-3-3-3. Combination treatments resulted in total disruption of microtubules and depletion of putrescine, spermidine and spermine. In addition, phosphorylation of Akt and nuclear localization of cyclin D1 were altered by 2ME/BE-3-3-3 combination. Our results suggest an important strategy to induce apoptosis of breast cancer cells, with potential applications in therapy.

CR011, a fully human monoclonal antibody-auristatin E conjugate, for the treatment of melanoma.

PURPOSE: Advanced melanoma is a highly drug-refractory neoplasm representing a significant unmet medical need. We sought to identify melanoma-associated cell surface molecules and to develop as well as preclinically test immunotherapeutic reagents designed to exploit such targets. EXPERIMENTAL DESIGN AND RESULTS: By transcript profiling, we identified glycoprotein NMB (GPNMB) as a gene that is expressed by most metastatic melanoma samples examined. GPNMB is predicted to be a transmembrane protein, thus making it a potential immunotherapeutic target in the treatment of this disease. A fully human monoclonal antibody, designated CR011, was generated to the extracellular domain of GPNMB and characterized for growth-inhibitory activity against melanoma. The CR011 monoclonal antibody showed surface staining of most melanoma cell lines by flow cytometry and reacted with a majority of metastatic melanoma specimens by immunohistochemistry. CR011 alone did not inhibit the growth of melanoma cells. However, when linked to the cytotoxic agent monomethylauristatin E (MMAE) to generate the CR011-vcMMAE antibody-drug conjugate, this reagent now potently and specifically inhibited the growth of GPNMB-positive melanoma cells in vitro. Ectopic overexpression and small interfering RNA transfection studies showed that GPNMB expression is both necessary and sufficient for sensitivity to low concentrations of CR011-vcMMAE. In a melanoma xenograft model, CR011-vcMMAE induced significant dose-proportional antitumor effects, including complete regressions, at doses as low as 1.25 mg/kg. CONCLUSION: These preclinical results support the continued evaluation of CR011-vcMMAE for the treatment of melanoma.

Application of single molecule technology to rapidly map long DNA and study the conformation of stretched DNA.

Herein we describe the first application of direct linear analysis (DLA) to the mapping of a bacterial artificial chromosome (BAC), specifically the 185.1 kb-long BAC 12M9. DLA is a single molecule mapping technology, based on microfluidic elongation and interrogation of individual DNA molecules, sequence-specifically tagged with bisPNAs. A DNA map with S/N ratio sufficiently high to detect all major binding sites was obtained using only 200 molecule traces. A new method was developed to extract an oriented map from an averaged map that included a mixture of head-first and tail-first DNA traces. In addition, we applied DLA to study the conformation and tagging of highly stretched DNA. Optimal conditions for promoting sequence-specific binding of bisPNA to an 8 bp target site were elucidated using DLA, which proved superior to electromobility shift assays. DLA was highly reproducible with a hybridized tag position localized with an accuracy of +/-0.7 microm or +/-2.1 kb demonstrating its utility for rapid mapping of large DNA at the single molecule level. Within this accuracy, DNA molecules, stretched to at least 85% of their contour length, were stretched uniformly, so that the map expressed in relative coordinates, was the same regardless of the molecule extension.

Single DNA molecule stretching in sudden mixed shear and elongational microflows.

High-throughput stretching and monitoring of single DNA molecules in continuous elongational flow offers compelling advantages for biotechnology applications such as DNA mapping. However, the polymer dynamics in common microfluidic implementations are typically complicated by shear interactions. These effects were investigated by observation of fluorescently labeled 185 kb bacterial artificial chromosomes in sudden mixed shear and elongational microflows generated in funneled microfluidic channels. The extension of individual free DNA molecules was studied as a function of accumulated fluid strain and strain rate. Under constant or gradually changing strain rate conditions, stretching by the sudden elongational component proceeded as previously described for an ideal elongational flow (T. T. Perkins, D. E. Smith and S. Chu, Science, 1997, 276, 2016): first, increased accumulated fluid strain and increased strain rate produced higher stretching efficiencies, despite the complications of shear interactions; and second, the results were consistent with unstretched molecules predominantly in hairpin conformations. More abrupt strain rate profiles did not deliver a uniform population of highly extended molecules, highlighting the importance of balance between shear and elongational components in the microfluidic environment for DNA stretching applications. DNA sizing with up to 10% resolution was demonstrated. Overall, the device delivered 1000 stretched DNA molecules per minute in a method compatible with diffraction-limited optical sequence motif mapping and without requiring laborious chemical modifications of the DNA or the chip surface. Thus, the method is especially well suited for genetic characterization of DNA mixtures such as in pathogen fingerprinting amidst high levels of background DNA.

Bending of the estrogen response element by polyamines and estrogen receptors alpha and beta: a fluorescence resonance energy transfer study.

Estrogenic regulation of gene expression is mediated by the binding of the hormone to its receptors (ERalpha and ERbeta) followed by their binding to estrogen response element (ERE). Previous studies showed that natural polyamines -- putrescine, spermidine, and spermine -- facilitated ERalpha.ERE recognition. We determined the effects of natural and synthetic polyamines on the bending of a 27-mer oligonucleotide (ODN) harboring the ERE (ERE-ODN), using fluorescence resonance energy transfer (FRET) technique. Complementary strands of the ERE-ODN were labeled with fluorescein and tetramethylrhodamine, as donor and acceptor, respectively. The ERE-ODN was intrinsically bent with an end-to-end distance of 76 +/- 2 Angstrom, compared to a theoretical value of 98 Angstrom. The end-to-end distance of the ERE-ODN was reduced to 64 Angstrom in the presence of 250 microM spermine. A control ODN with scrambled sequence did not show intrinsic bending or spermine-induced bending. Alkyl substitution at the pendant amino groups reduced the ability of spermine to bend the ERE-ODN. Both ERalpha and ERbeta decreased the end-to-end distance of the ERE-ODN, although ERalpha was more efficient than ERbeta in inducing ERE bending. Spermine-induced bending of the ERE-ODN was significantly increased by ERalpha. Fluorescence anisotropy measurement showed that the equilibrium association constant of ERalpha-ERE binding increased by 12-fold in the presence of 250 microM spermine compared to control. The free energy change (Delta G) of ERalpha.ERE complex formation was -13.1 kcal/mol at 22 degrees C in the presence of spermine. Our results suggest that polyamine-induced bending of the ERE might be a mechanism for enhancing ERalpha-ERE binding affinity and thereby fine-tuning the transcriptional response of estrogen-responsive genes.

ADAM17 Inhibitors Attenuate Corneal Epithelial Detachment Induced by Mustard Exposure.

PURPOSE: Sulfur mustard, nitrogen mustard (NM), and 2-chloroethyl ethyl sulfide all cause corneal injury with epithelial-stromal separation, differing only by degree. Injury can resolve in a few weeks or develop into chronic corneal problems. These vesicants induce microbullae at the epithelial-stromal junction, which is partially caused by cleavage of transmembranous hemidesmosomal collagen XVII, a component anchoring the epithelium to the stroma. ADAM17 is an enzyme involved in wound healing and is able to cleave collagen XVII. The activity of ADAM17 was inhibited in vesicant-exposed corneas by four different hydroxamates, to evaluate their therapeutic potential when applied 2 hours after exposure, thereby allowing ADAM17 to perform its early steps in wound healing. METHODS: Rabbit corneal organ cultures exposed to NM for 2 hours were washed, then incubated at 37°C for 22 hours, with or without one of the four hydroxamates (dose range, 0.3-100 nmol in 20 µL, applied four times). Corneas were analyzed by light and immunofluorescence microscopy, and ADAM17 activity assays. RESULTS: Nitrogen mustard-induced corneal injury showed significant activation of ADAM17 levels accompanying epithelial-stromal detachment. Corneas treated with hydroxamates starting 2 hours post exposure showed a dose-dependent ADAM17 activity inhibition up to concentrations of 3 nmol. Of the four hydroxamates, NDH4417 (N-octyl-N-hydroxy-2-[4-hydroxy-3-methoxyphenyl] acetamide) was most effective for inhibiting ADAM17 and retaining epithelial-stromal attachment. CONCLUSIONS: Mustard exposure leads to corneal epithelial sloughing caused, in part, by the activation of ADAM17 at the epithelial-stromal junction. Select hydroxamate compounds applied 2 hours after NM exposure mitigated epithelial-stromal separation.

Differential effects of 16alpha-hydroxyestrone and 2-methoxyestradiol on cyclin D1 involving the transcription factor ATF-2 in MCF-7 breast cancer cells.

We studied the effects of 2-methoxyestradiol (2-ME2) and 16alpha-hydroxyestrone (16alpha-OHE1), two metabolites of estradiol (E2), on DNA synthesis, cell cycle progression and cyclin D1 protein levels in estrogen receptor-positive MCF-7 cells. E2 and 16alpha-OHE1 stimulated DNA synthesis, and 2-ME2 inhibited the stimulatory effects of these agents. E2 and 16alpha-OHE1 stimulated the progression of cells from G1 to S phase and this effect was attenuated by 2-ME2. Western blot analysis showed that E2 and 16alpha-OHE1 increased cyclin D1 protein level by about fourfold compared with control. 2-ME2 had no significant effect on cyclin D1; however, it prevented the accumulation of cyclin D1 in the presence of E2 and 16alpha-OHE1. Cells transfected with a cyclin D1 reporter gene and treated with E2 or 16alpha-OHE1 showed 7- and 9.5-fold increase respectively in promoter activity compared with control. This activity was significantly inhibited by 2-ME2. Cyclin D1 transactivation was mediated by the cAMP response element (CRE) region, which binds activating transcription factor 2 (ATF-2). DNA affinity assay showed 2.5- and 3.5-fold increases in ATF-2 binding to CRE in the presence of E2 and 16alpha-OHE1 respectively. The binding of ATF-2 was inhibited by the presence of 2-ME2. These results show that 2-ME2 can downregulate cyclin D1 and thereby cell cycle progression by a mechanism involving the disruption of ATF-2 binding to cyclin D1 promoter.

Activation of cyclin D1 by estradiol and spermine in MCF-7 breast cancer cells: a mechanism involving the p38 MAP kinase and phosphorylation of ATF-2.

Estradiol (E2) and the naturally occurring polyamines (putrescine, spermidine, and spermine) play important roles in breast cancer cell growth and differentiation. We examined the effects of E2 and spermine on the phosphorylation and DNA binding of activating transcription factor-2 (ATF-2) in MCF-7 breast cancer cells. ATF-2 is a transcription factor involved in estrogenic regulation of cyclin D1 gene, and thereby cell cycle progression. DNA affinity immunoblot assays showed a six- to eightfold increase in the binding of ATF-2 to a 74-mer ATF/CRE oligonucleotide (ODN1) from cyclin D1 promoter in the presence of 4 nM E2 and 0.5 mM spermine, compared to untreated control. Individual treatments with E2 or spermine caused a twofold or lower increase in ATF-2 binding to ODN1. Immunoblotting with phospho-ATF-2 antibody showed that increased DNA binding of ATF-2 was associated with its phosphorylation. A p38 MAP kinase inhibitor, PD169316, inhibited ATF-2 phosphorylation. In contrast, the MEK-ERK1/2 inhibitor, PD98059, or the JNK inhibitor, SP600125, had no significant effect on DNA binding of ATF-2. Cyclin D1 promoter (-1745CD1) activity increased by approximately 12-fold (above control) in the presence of E2 and spermine, compared to a sixfold increase in the presence of E2 alone and a twofold increase with spermine. Cells transfected with a dominant negative mutant of ATF-2 showed decreased transactivation of cyclin D1 promoter in response to E2 and spermine. These results indicate that spermine can enhance E2-induced cell signaling and cyclin D1 transcription by activation of the p38 MAP kinase and phosphorylation of ATF-2, contributing to breast cancer cell proliferation.

Bis(ethyl)norspermine potentiates the apoptotic activity of the pure antiestrogen ICI 182780 in breast cancer cells.

We studied the effects of ICI 182780 and bis(ethyl)norspermine (BE-3-3-3) on cell growth and apoptosis of estrogen receptor-positive MCF-7 breast cancer cells. Combination treatment with 100 nM ICI 182780 and 5 microM BE-3-3-3 for 6 days inhibited cell growth by 74.3+/-8.4% in MCF-7 cells, compared to that of 25.4+/-5.8 and 45.8+/-12.2%, respectively, when ICI 182780 and BE-3-3-3 were used as single agents. Treatment with 100 nM ICI 182780 and 5 microM BE-3-3-3 as single agents resulted in 9.1+/-1.0% and 35.1+/-4.5% apoptosis, respectively, as measured by APO-BRDU assay. When ICI 182780 and BE-3-3-3 were used in combination, the percentage of apoptosis was 60.6+/-3.8%. Improved efficacy of ICI 182780 and BE-3-3-3 combination on growth inhibition was observed for T-47D cells also. Western blot analysis showed that combinations of ICI 182780 and BE-3-3-3 caused down-regulation of the anti-apoptotic Bcl-2 and Bcl-XL proteins and increased the level of the pro-apoptotic Bax protein. Combination treatment also increased caspase-8 activation. Analysis of polyamine levels 48 h after combination treatment showed that spermidine and spermine levels were down regulated significantly. These studies indicate a potentially effective combination strategy for breast cancer treatment. Our results also link the down-regulation of polyamine pathway to apoptotic cell death and regulation of mediators of cell death.

Estrogen receptors as targets for drug development for breast cancer, osteoporosis and cardiovascular diseases.

Estrogen receptors (ERs) are proteins that mediate the action of estradiol and a series of natural and synthetic chemicals that mimic the estradiol structure. Estrogenic action was initially attributed to a single type of ER, now known as ERalpha, but ERbeta was discovered in 1995. Tissue specific distribution and the intensity of expression of these proteins determine the first response of tissues to estrogenic compounds. Estrogens and ERs play a major role in the origin and progression of breast cancer, and antiestrogens that block ER function are useful for breast cancer prevention and treatment. Estrogen mimetics, however, do not fall into distinct categories of agonists and antagonists, since their action is regulated by tissue-specific expression of a number of auxiliary proteins called coactivators or corepressors. In addition, small molecules such as polyamines, fattyacids, and thioredoxin may modulate ER function. Estrogenic functions encompass multiple organ systems, including the reproductive, skeletal, cardiovascular, and nervous system. Estrogens are critical for bone remodeling and mineralization so that estrogen replacement therapy is proven to strengthen bone health in post-menopausal women. Ideally, selective blockade of ER function in breast epithelial cells should be accompanied by growth support on bone and cardiovascular systems. The details of estrogenic function in different organs are to be fully realized, in order to better utilize selective estrogen receptor modulators (SERMs) to fight not only breast cancer but also osteoporosis and cardiovascular diseases. Current research on SERMs points toward accomplishing this goal by exploiting ER as a versatile target against multiple diseases.

A novel method of Multiplexed Competitive Antibody Binning for the characterization of monoclonal antibodies.

We have developed a novel method of high-throughput Multiplexed Competitive Antibody Binning (MCAB). Using only a small amount of antibody and antigen, this method enables the sorting of a large, complex panel of monoclonal antibodies into different bins based on cross-competition for antigen binding. The MCAB assay builds on Luminex multiplexing bead-based technology to detect antibody competition. Because of its high sensitivity, the MCAB method is immediately applicable after identification of antigen-positive mAbs, providing information useful for advancing mAb candidates into further testing. The MCAB assay also can be used for sorting mAbs into binding groups after screening for functional activity.

Summary of the National Toxicology Program's report of the endocrine disruptors low-dose peer review.

At the request of the U.S. Environmental Protection Agency (U.S. EPA), the National Toxicology Program organized an independent and open peer review to evaluate the scientific evidence on low-dose effects and nonmonotonic dose-response relationships for endocrine-disrupting chemicals in mammalian species. For this peer review, "low-dose effects" referred to biologic changes that occur in the range of human exposures or at doses lower than those typically used in the standard testing paradigm of the U.S. EPA for evaluating reproductive and developmental toxicity. The demonstration that an effect is adverse was not required because in many cases the long-term health consequences of altered endocrine function during development have not been fully characterized. A unique aspect of this peer review was the willing submission of individual animal data by principal investigators of primary research groups active in this field and the independent statistical reanalyses of selected parameters prior to the peer review meeting by a subpanel of statisticians. The expert peer-review panel (the panel) also considered mechanistic data that might influence the plausibility of low-dose effects and identified study design issues or other biologic factors that might account for differences in reported outcomes among studies. The panel found that low-dose effects, as defined for this review, have been demonstrated in laboratory animals exposed to certain endocrine-active agents. In some cases where low-dose effects have been reported, the findings have not been replicated. The shape of the dose-response curves for reported effects varied with the end point and dosing regimen and were low-dose linear, threshold-appearing, or nonmonotonic. The findings of the panel indicate that the current testing paradigm used for assessments of reproductive and developmental toxicity should be revisited to see whether changes are needed regarding dose selection, animal-model selection, age when animals are evaluated, and the end points being measured following exposure to endocrine-active agents.