DNA aneuploidy as a specific marker of neoplastic cells in FNAB of the thyroid

OBJECTIVE: To investigate whether DNA image cytometry (ICM) could be of value in the specific identification of neoplastic cells in cytologic specimens of the thyroid. STUDY DESIGN: FNABs of thyroid from 162 patients with different benign and neoplastic diseases were investigated. Nuclear DNA content in thyroid cells was measured after Feulgen staining using a TV image analysis system. Data were correlated with clinical and histologic patient follow-up. The occurrence of abnormal DNA stemlines was used as a marker for aneuploidy and thus for neoplasia. RESULTS: None of the 89 cases without tumor cells revealed DNA aneuploidy. An abnormal DNA stemline was found in 55% of histologically proven benign thyroid tumors (follicular or oncocytic adenomas) and in 59.5% of malignant tumors. The positive predictive value of DNA aneuploidy in FNABs of the thyroid for neoplasms was 100%. The negative predictive value of DNA nonaneuploidy for the prediction of tumor-free histologic or clinical follow-up was 79.4%. CONCLUSION: DNA image cytometry may be helpful in the specific identification of neoplastic follicular cells. DNA ICM on FNABs of the thyroid is an additional tool to achieve early identification of patients with nodular lesions of the thyroid that have to be operated on. DNA euploidy excludes the presence of neither malignancy nor neoplasia.     

Dendritic cell immunotherapy induces antitumour response in parathyroid carcinoma and neuroendocrine pancreas carcinoma.

Parathyroid carcinomas and neuroendocrine carcinomas of the pancreas are rare malignancies in humans. Because of their low radio- and chemosensibility, they fail to respond to conventional therapy. We therefore tested a dendritic cell immunotherapy in an attempt to control the tumour growth in two patients. Studies on mice and humans have demonstrated the potent capacity of dendritic cells to induce specific antitumour immunity. Mature dendritic cells were generated from peripheral blood monocytes in the presence of granulocyte/macrophage colony-stimulating factor, interleukin 4 and tumour necrosis factor alpha. Dendritic cells were either loaded with parathyroid hormone (PTH) or with (pancreas) tumour-derived lysate (TL), respectively, and were delivered by subcutaneous injections. All immunizations were well tolerated with no side effects, and were administered on an outpatient basis. After repeated vaccinations, specific in vivo immune response was demonstrated by positive delayed-type hypersensitivity (DTH) toward PTH or TL, demonstrating the efficient generation of antigen-specific memory T-cells. DTH reactivity was accompanied by a significant decrease of tumour markers in both patients. This approach might be generally applicable to other advanced, radio- and chemotherapy-resistant endocrine malignancies.     

Effect of fruit maturity,seed weight and storage time on the viability and germination of the seed of candelilla (<i>Euphorbia antisiphylitica</i> Zucc.)

Candelilla (Euphorbia antisiphylitica Zucc.) is a very important plant resource in the arid lands of Northern Mexico. This is because the wax content coating the stem has unique properties which have been useful for multiple applications in the food industry, electronics, cosmetics, etc. However, the intensive exploitation of this resource has caused a great decrease in the populations of this species making necessary to consider strategies for their conservation and sustainable use. One of the primary needs with regeneration purposes is to know their reproductive processes, particularly the biotic and/or abiotic factors that determine the viability and germination of seeds. The present study evaluated the (1) germination and seed viability in relation to the ripeness degree of the fruit at the time of collection, (2) weight of the seed (low, medium and high), and (3) storage time (1, 3, and 5 months). Fruits from four locations, two in the State of Coahuila (Las Coloradas and Candela) and two in the State of Nuevo Leon (Icamole 1 and Icamole 2), were collected. Three germination assays were carried out corresponding to each month of storage. Seed viability was determined by the tetrazolium test. The average weight of the candelilla seeds was 0.0029 ± 0.0010 g, with extreme average values of 0.0018 ± 0.0006 g at Las Coloradas and 0.0036 ± 0.0010 g in Icamole 2. Those seeds with heavier weight obtained from red fruits and with 1 month of storage showed the highest average percentage of viability (66.87 ± 24.19%). At the same time, seeds with around average weight, obtained from red fruits and five months of storage, showed the highest average germination percentage (50.00 ± 9.42%).     

Nifedipine loaded chitosan microspheres: characterization of internal structure

In the present investigation, a simple technique was employed to obtain cross-sections of unloaded and nifedipine loaded chitosan microspheres. Microspheres, adhering to a polymerized resin block, were cut with an ultramicrotome and viewed with a scanning electron microscope. Unloaded microspheres exhibited a uniform dense matrix structure while crystals of nifedipine were clearly visible in the drug-loaded microspheres. At 2% drug loading, however, no crystals could be seen in the microspheres indicating that either the drug was molecularly dispersed or dissolved in the matrix at this concentration. This was confirmed by powder X-ray diffractometry studies where no peak due to crystalline nifedipine was observed. At high Span 85 concentration (1.5% w/v), the external surface of the microspheres collapsed, but the internal structure remained dense. When the drug was dispersed in the chitosan solution with stirring during preparation, the entrapment was good and the shape of the crystals was changed. The internal structure of the microspheres following dissolution exhibited the presence of pores.     

Case-control study of self reported genitourinary infections and risk of gastroschisis: findings from the national birth defects prevention study, 1997-2003

Objective To assess the association between genitourinary infections in the month before conception to the end of the first trimesterand gastroschisis.Design Case-control study with self reported infections from a computer assisted telephone interview.Setting National birth defects prevention study, a multisite, population based study including 10 surveillance systems for birth defects in the United States.Participants Mothers of 505 offspring with gastroschisis and 4924 healthy liveborn infants as controls.Main outcome measure Adjusted odds ratios for gastroschisis with 95% confidence intervals.Results About 16% (n=81) of case mothers and 9% (n=425) of control mothers reported a genitourinary infection in the relevant time period; 4% (n=21) and 2% (n=98) reported a sexually transmitted infection and 13% (n=67) and 7% (n=338) reported a urinary tract infection, respectively. Case mothers aged <25 years reported higher rates of urinary tract infection alone and in combination with a sexually transmitted infection compared with control mothers. In women who reported both types of infection, there was a greater risk of gastroschisis in offspring (adjusted odds ratio 4.0, 95% confidence interval 1.4 to 11.6).Conclusion There is a significant association between self reported urinary tract infection plus sexually transmitted infection just before conception and in early pregnancy and gastroschisis.     

Dissecting the binding energy epitope of a high-affinity variant of human growth hormone: cooperative and additive effects from combining mutations from independently selected phage display mutagenesis libraries

Phage display mutagenesis is a widely used approach to engineering novel protein properties and is especially powerful in probing structure-function relationships in molecular recognition processes. The relative contributions of additive and cooperative binding forces and the influence of conformational diversity in producing a novel protein-protein interface is investigated using as a model an ultra-high-affinity receptor binding variant of human growth hormone (hGHv) that has been previously affinity matured. The modular aspect of how the mutations were grouped in the phage display libraries and combined allowed for a systematic probing of the inherent functional cross-talk between the different secondary structure elements that make up the remodeled hGHv binding surface. We performed an alanine scanning analyses of 35 hGHv residues and determined the kinetics of each variant by surface plasmon resonance (SPR). This analysis showed that there is a significant difference between the additive and cooperative binding forces existing among the selected residues in each library module, and the binding advantage of these residues is maximized over the original wild-type residue when in the context of the other mutations in the library. The degree to which residues in a particular mutagenesis library display binding cooperativity characteristics is generally correlated with the conformational plasticity of the polypeptide chain. Additionally, these cooperativity effects change when the mutations from one library are combined with the mutations from one or several of the other separate libraries. This supports the idea that significant functional cross-talk exists between the combined library modules that can affect the binding energetics of individual residues over a large distance.     

Correlation between thyroid hormone status and hepatic hyperplasia and hypertrophy caused by the peroxisome proliferator-activated receptor alpha agonist Wy-14,643

BACKGROUND: The metabolic inhibitor rotenone inhibits hepatocellular proliferation and the incidence of liver cancer resulting from exposure to the PPARalpha agonist Wy-14,643, via unknown mechanisms. Since the absence of thyroid hormones diminishes hepatomegaly, an early biomarker for the hepatocarcinogenicity induced by PPARalpha agonists, this study was undertaken to investigate whether rotenone might interference with the ability of Wy-14,643 to alter the animal thyroid status. METHODS: Male B6C3F1 mice were given Wy-14,643 (100 ppm), rotenone (600 ppm) or a mixture of both, in the feed for 7 days. Bromodeoxyuridine (BrDU), marker of cell replication, was delivered through subcutaneously implanted osmotic mini-pumps. At the end of the experiment, sera were collected and corticosterone and thyroid hormone levels were measured by solid-phase radioimmunoassay kits. In addition, liver tissue samples were stained immunohistochemically for BrDU to determine percentages of labeled cells. Further, cell surface area was determined from images generated by a Zeiss Axioplan microscope equipped with a plan Neofluar x40 0.75 na objective. Tracings of individual hepatocyte perimeters were then analyzed and cell-surface areas were calculated using MicroMeasure FL-4000. RESULTS: Wy-14,643 caused a significant increase in liver weights, hepatocyte BrDU labeling index (LI), and hepatocyte surface area. In animals which received both Wy-14,643 and rotenone simultaneously, all of these effects were significantly less pronounced compared with mice that received Wy-14,643 alone. Rotenone alone decreased liver weights, LI and surface area. The Free Thyroid Index (FTI), which provides an accurate reflection of the animal's thyroid status, was 5.0 +/- 0.3 in control mice. In animals exposed to rotenone, these values decreased to 2.0 +/- 0.9, but in animals which received Wy-14,643, levels increased significantly to 7.7 +/- 0.9. FTI values decreased to 3.4 +/- 0.8 in mice receiving both rotenone and Wy-14,643. CONCLUSION: A strong correlation was observed between the animal thyroid status and both, hepatocyte proliferation (r2 = 0.62), and hepatocyte surface area (r2 = 0.83). These results support the hypothesis that the thyroid status of the animal plays a role in PPARalpha-induced hepatocellular proliferation and liver cell enlargement. Both these events are known to contribute to the expression of liver cancer in response to the activation of PPARalpha.     

A high-throughput fluorescence polarization anisotropy assay for the 70N domain of replication protein A

Replication protein A (RPA) interacts with multiple checkpoint proteins and promotes signaling through the ATR kinase, a key regulator of checkpoint pathways in the mammalian response to DNA damage. In cancer cells, increased DNA repair activity contributes to resistance to chemotherapy. Therefore, small molecules that block binding of checkpoint proteins to RPA may inhibit the DNA damage response and, thus, sensitize cancer cells to DNA-damaging agents. Here we report on the development of a homogeneous, high-throughput fluorescence polarization assay for identifying compounds that block the critical protein-protein interaction site in the basic cleft of the 70N domain of RPA (RPA70N). A fluorescein isothiocyanate (FITC)-labeled peptide derived from the ATR cofactor, ATRIP, was used as a probe in the binding assay. The ability of the assay to accurately detect relevant ligands was confirmed using peptides derived from ATRIP, RAD9, MRE11, and p53. The assay was validated for use in high-throughput screening using the Spectrum collection of 2000 compounds. The FPA assay was performed with a Z' factor of ≥ 0.76 in a 384-well format and identified several compounds capable of inhibiting the RPA70N binding interface.