Alterations in Cell Motility, Proliferation, and Metabolism in Novel Models of Acquired Temozolomide Resistant Glioblastoma.

Glioblastoma (GBM) is an aggressive and incurable tumor of the brain with limited treatment options. Current first-line standard of care is the DNA alkylating agent temozolomide (TMZ), but this treatment strategy adds only ~4 months to median survival due to the rapid development of resistance. While some mechanisms of TMZ resistance have been identified, they are not fully understood. There are few effective strategies to manage therapy resistant GBM, and we lack diverse preclinical models of acquired TMZ resistance in which to test therapeutic strategies on TMZ resistant GBM. In this study, we create and characterize two new GBM cell lines resistant to TMZ in vitro, based on the 8MGBA and 42MGBA cell lines. Analysis of the TMZ resistant (TMZres) variants in conjunction with their parental, sensitive cell lines shows that acquisition of TMZ resistance is accompanied by broad phenotypic changes, including increased proliferation, migration, chromosomal aberrations, and secretion of cytosolic lipids. Importantly, each TMZ resistant model captures a different facet of the "go" (8MGBA-TMZres) or "grow" (42MGBA-TMZres) hypothesis of GBM behavior. These in vitro model systems will be important additions to the available tools for investigators seeking to define molecular mechanisms of acquired TMZ resistance.

Frequent loss of the BLID gene in early-onset breast cancer.

The BH3-like motif-containing inducer of cell death (BLID) is an intronless gene localized on 11q24.1. Loss of that region has frequently been reported in early-onset breast cancer and is significantly associated with poor prognosis and reduced survival. Downregulation of BLID is associated with younger age, triple-negative phenotype, and reduced disease-free and overall survival of breast cancer patients. In this study, we investigated allelic loss of BLID in breast tumor specimens from 78 women with invasive breast cancer using 2 dinucleotide polymorphic markers closely linked to the BLID gene (no intragenic marker for BLID is available). Seventy-three cases were informative. Overall, loss of heterozygosity (LOH) at the BLID locus was detected in 32% of the informative cases (23/73). However, in patients 40 years old and younger, LOH was detected in 50% of the cases (9/18). Patients aged 40 years and younger were significantly more likely to experience LOH than those aged 41-55 years (p = 0.04). Specifically, the odds of BLID loss for patients aged 40 years and younger were 3.7 times the odds of loss for patients aged 41-55 years (95% CI, 1.1-13). Our findings suggest a tumor suppressor role of the BLID gene in early-onset breast cancer.

Subcutaneous scalp emphysema, pneumo-orbitis and pneumocephalus in a neonate on high humidity high flow nasal cannula.

High humidity high flow nasal cannula has become a widely used alternative for nasal continuous positive airway pressure for the treatment of apnea of prematurity. We describe our experience of one incident of subcutaneous scalp emphysema, pneumo-orbitis and pneumocephalus with concomitant use of the high-flow nasal cannula.

Patterns of DNA copy number changes in sentinel lymph node breast cancer metastases.

The sentinel lymph node (SLN) is considered to be the first axillary node that contains malignant cells in metastatic breast tumors, and its positivity is currently used in clinical practice as an indication for axillary lymph node dissection. Therefore, accurate evaluation of the SLN for the presence of breast metastatic cells is essential. The main aim of our study is to characterize the genomic changes present in the SLN metastatic samples with the ultimate goal of improving the predictive value of SLN evaluation. Twenty paired samples of SLN metastases and their corresponding primary breast tumors (PBT) were investigated for DNA copy number changes using comparative genomic hybridization (CGH). Non-random DNA copy number changes were observed in all the lesions analyzed, with gains being more common than losses. In 75% of the cases there was at least one change common to both PBT and SLN. The most frequent changes detected in both lesions were gains of 1pter-->p32, 16, 17, 19, and 20 and losses of 6q13-->q23 and 13q13-->q32. In the PBT group, alterations on chromosomes 1, 16, and 20 were the most frequent, whereas chromosomes 1, 6, and 19 were the ones with the highest number of changes in the SLN metastatic group. A positive correlation was found between the DNA copy number changes per chromosome in each of the groups. Our findings indicate the presence of significant DNA copy number changes in the SLN metastatic lesions that could be used in the future as additional markers to improve the predictive value of SLN biopsy procedure.

Evaluation of paediatric osteosarcomas by classic cytogenetic and CGH analyses.

Classic cytogenetic and comparative genomic hybridisation (CGH) data on osteosarcomas have been reported extensively in the literature. However, the number of paediatric osteosarcoma cases studied below the age of 14 years remains relatively small. This study reports four new cases of paediatric osteosarcoma in patients aged 3 to 13 years, evaluated by classic cytogenetics and CGH analyses. Clonal chromosomal alterations were detected in all the cases and included structural rearrangements at 1p11-13, 1q11, 4q27-33, 6p23-25, 6q16-25, 7p13-22, 7q11-36, 11p10-15, 11q23, 17p11.2-13, 21p11, and 21q11-22. The CGH analysis revealed recurrent gains at 1p, 4q, 17p, and 21q and losses at 3q and 16p. Five amplification sites were observed at 1q11-23, 6p21, 8q13, 8q21.3-24.2, and 17p. The data are discussed and compared with other cytogenetic reports in the literature.

Peripheral-type benzodiazepine receptor levels correlate with the ability of human breast cancer MDA-MB-231 cell line to grow in SCID mice.

MDA-MB-231 (MDA-231) human breast cancer cells have a high proliferation rate, lack the estrogen receptor, express the intermediate filament vimentin, the hyaluronan receptor CD44, and are able to form tumors in nude mice. The MDA-231 cell line has been used in our laboratory to examine the role of the peripheral-type benzodiazepine receptor (PBR) in the progression of cancer. During these studies 2 populations of MDA-231 cells were subcloned based on the levels of PBR. The subclones proliferated at approximately the same rate, lacked the estrogen receptor, expressed vimentin and CD44, and had the same in vitro chemoinvasive and chemotactic potential. Both restriction fragment length polymorphism and comparative genomic hybridization analyses of genomic DNA from these cells indicated that both subclones are of the same genetic lineage. Only the subclone with high PBR levels, however, was able to form tumors when injected in SCID mice. These data suggest that the ability of MDA-231 cells to form tumors in vivo may depend on the amount of PBR present in the cells.

Peptide T blocks GP120/CCR5 chemokine receptor-mediated chemotaxis.

We previously reported that certain short gp120 V2 region peptides homologous to vasaoactive intestinal peptide (VIP), such as "peptide T," were potent inhibitors of gp120 binding, infectivity, and neurotoxicity. The present study shows that synthetic V2-region-derived peptides have potent intrinsic chemotaxis agonist activity for human monocytes and also act as antagonists of high-affinity (0.1 pM) gp120-mediated monocyte chemotaxis. Selectivity is shown in that peptide T is more potent at suppressing M-tropic than T-tropic gp120 chemotaxis. Peptide T was also able to suppress monocyte chemotaxis to MIP-1beta, a chemokine with selectivity for CCR5 chemokine receptors, while chemotaxis of the more promiscuous ligand RANTES was not inhibited, nor was chemotaxis mediated by SDF-1alpha. In order to determine if peptide T mediated its gp120 antagonistic effects via modulation of CCR5 receptors, RANTES chemotaxis was studied using a CCR5 receptor-transfected HOS cell line. In this case, RANTES chemotaxis was potently inhibited by V2-region-derived short peptides. Peptide T also partially suppressed (125)I-MIP1-beta binding to human monocytes, suggesting action at a subset of MIP1-beta receptors. The V2 region of gp120 thus contains a potent receptor binding domain and synthetic peptides derived from this region modulate CCR5 chemokine receptor chemotactic signaling caused by either gp120 or chemokine ligands. The results have therapeutic implications and may explain recent clinical improvements, in that HIV/gp120 actions at CCR5 receptors, such as occur in the brain or early infection, would be susceptible to peptide T inhibition.

Effect of pregnancy-specific beta 1-glycoprotein on the development of preimplantation embryo.

The objective of this study is to test the hypothesis that members of the pregnancy-specific beta 1-glycoprotein (PSG) family enhance the growth and maturation of embryos. cDNA encoding two members of the PSG family, namely PSG1 and PSG3, were expressed in Chinese Hamster Ovary (CHO) cells with the expression vector pH beta APr-1-neo. Two-cell stage mouse embryos were co-cultured in a two-chamber system with CHO cells expressing either recombinant PSG1 (rPSG1) or PSG3 (rPSG3) in the presence and absence of neutralizing PSG antibodies. The cleavage and maturation stage of the embryos was assessed at 12-hr intervals. Mouse embryos co-cultured with transfectants expressing rPSG1 showed a significant enhancement of cleavage and maturation rate compared to controls with P < 0.005-0.004. In co-cultures with CHO cells expressing rPSG3, no significant difference from the controls was observed in the early stage of development until late blastocyst formation. At that stage, there was a statistically significant enhancement of development by rPSG3 when compared to controls with P < 0.001. These results suggest that PSG1 and PSG3 exhibit embryotropic activity at different stages of development in the mouse model.

Papillary thyroid carcinoma, parathyroid adenoma, and unexplained hypercalcitoninemia: an unusual presentation of multiple endocrine neoplasia type 2A?

Multiple endocrine neoplasia type 2 (MEN 2) is a rare syndrome of medullary thyroid carcinoma (MTC) with pheochromocytoma and/or primary hyperparathyroidism (PHP), usually due to multigland hyperplasia. MEN 2 is associated with several RET protooncogene mutations. A 61-year-old woman with a family history of RET-positive MTC presented with a solitary thyroid nodule. Fine-needle aspiration biopsy was suspicious for neoplasm. Biochemical studies revealed basal hypercalcitoninemia (116 pg/mL [normal <26]) and PHP (serum calcium, 10.9 mg/dL; intact PTH, 113.2 pg/mL [10.0-65.0]). Pheochromocytoma screening was negative. A provisional diagnosis of MEN 2 was made, but at surgery, a single parathyroid adenoma was resected and frozen sections of several lymph nodes revealed papillary thyroid carcinoma (PTC). A total thyroidectomy was performed. Final histological diagnosis was PTC and parathyroid adenoma with no evidence of MTC. Postoperatively, RET mutation testing was positive. The basal calcitonin (CT) fell to 25 pg/mL, but peaked at 935 (normal <105) after pentagastrin infusion, consistent with occult MTC. After radioiodine ablation, CT decreased further. Octreotide scanning was negative. Faced with PHP, a thyroid nodule, and a family history of MTC, clinicians tend to diagnose MEN 2. This patient had a single parathyroid adenoma and nonmedullary thyroid cancer, which the literature actually suggests to be an association more frequent than MEN 2. Yet, there remains compelling data in favor of occult MTC, leaving open the possibility of an MEN 2 variant with the rare association of PTC.

Neuropeptide Y: a novel angiogenic factor from the sympathetic nerves and endothelium.

Sympathetic nerves have long been suspected of trophic activity, but the nature of their angiogenic factor has not been determined. Neuropeptide Y (NPY), a sympathetic cotransmitter, is the most abundant peptide in the heart and the brain. It is released during nerve activation and ischemia and causes vasoconstriction and smooth muscle cell proliferation. Here we report the first evidence that NPY is angiogenic. At low physiological concentrations, in vitro, it promotes vessel sprouting and adhesion, migration, proliferation, and capillary tube formation by human endothelial cells. In vivo, in a murine angiogenic assay, NPY is angiogenic and is as potent as a basic fibroblast growth factor. The NPY action is specific and is mediated by Y1 and Y2 receptors. The expression of both receptors is upregulated during cell growth; however, Y2 appears to be the main NPY angiogenic receptor. Its upregulation parallels the NPY-induced capillary tube formation on reconstituted basement membrane (Matrigel); the Y2 agonist mimics the tube-forming activity of NPY, whereas the Y2 antagonist blocks it. Endothelium contains not only NPY receptors but also peptide itself, its mRNA, and the "NPY-converting enzyme" dipeptidyl peptidase IV (both protein and mRNA), which terminates the Y1 activity of NPY and cleaves the Tyr1-Pro2 from NPY to form an angiogenic Y2 agonist, NPY3-36. Endothelium is thus not only the site of action of NPY but also the origin of the autocrine NPY system, which, together with the sympathetic nerves, may be important in angiogenesis during tissue development and repair.

Ovarian angiogenesis in rabbits: endotheliotrophic chemoattractant activity from isolated follicles and dispersed granulosa cells.

To test the hypothesis that angiogenesis is an important variable in ovarian folliculogenesis, we measured endothelial cell migration (chemotaxis) in media conditioned by rabbit ovarian cells. Endothelial cell migration, a reliable predictor of angiogenesis in vivo, was stimulated by media conditioned by isolated intact follicles (0.4-2.2 mm in diameter) from either unstimulated or hCG-stimulated (pseudopregnant) rabbits. In separate experiments, endothelial cell migration was also stimulated by granulosa cell-conditioned media. Follicular chemoattractant activity was associated with a molecular weight greater than 30,000 but was not correlated with follicular size or steroid concentrations in the media, although there was no evidence to suggest that the biological activity detected in media conditioned by either intact follicles or dispersed granulosa cells was the same. Demonstration of nonsteroidal chemoattractant activity in media conditioned by intact follicles or by dispersed granulosa cells provides evidence that follicles secrete a vascular chemotactic factor, and is consistent with a role for angiogenesis in follicle growth.

The effect of endurance training on serum triiodothyronine kinetics in man: physical conditioning marked by enhanced thyroid hormone metabolism.

OBJECTIVE: We studied the relationship between endurance training, aerobic capacity, and T3 metabolism in healthy euthyroid men. DESIGN: T3 kinetic studies performed on two groups of subjects differentiated on the basis of physical activity status and aerobic capacity. SUBJECTS: Five endurance-trained athletes and five sedentary controls (mean +/- SD VO2 max = 48.2 +/- 7.1 vs 23.2 +/- 4.5 ml/kg/min, respectively) matched for age, body surface area, lean body mass, and baseline thyroid function. MEASUREMENTS: Kinetic analysis performed using serial serum T3 levels measured following oral T3 administration. Metabolic clearance rate, total volume of distribution, disposal rate, and total body pool calculated using non-compartmental analysis. RESULTS: When normalized for lean body mass, all kinetic parameters were 25-38% greater in the athletic group compared to controls (P < 0.05). Total volume of distribution, disposal rate, and total body pool were positively correlated with aerobic capacity (r = +0.69 to +0.79; P < 0.05). Metabolic clearance rate was positively correlated to a non-significant degree. CONCLUSIONS: These results confirm the findings of prior studies that thyroid hormone metabolism is altered by physical conditioning. In addition, we demonstrated a positive correlation between aerobic capacity and several parameters of T3 kinetics. Differences in absolute lean body mass cannot explain these findings; rather it appears that there is something qualitatively different in the way endurance-trained tissue processes thyroid hormone, compared to untrained tissue. The study was not designed to elucidate these differences at the cellular level; however, it does support a link between muscle physiology and T3 activity and may suggest a physiological role for thyroid hormone in physical conditioning.

Epidermal growth factor inhibits follicular response to human chorionic gonadotropin: possible role of cell to cell communication in the response to gonadotropin.

Epidermal growth factor (EGF) affects follicular steroidogenesis and expression of gonadotropin receptors. The effects of EGF on hCG-induced estradiol and progesterone secretion and ovulation were examined in the in vitro perfused rabbit ovary. We also examined the effects of EGF on hCG-induced progesterone secretion by isolated granulosa cells. In addition, distribution of hCG within the follicle was probed by immunohistochemical means 30 min after its administration to the in vitro perfused ovary. EGF significantly (P less than 0.05) reduced hCG-induced secretion of estradiol (control, 117 +/- 12 pg/min.follicle; 10 ng/ml EGF, 55 +/- 10) and progesterone (control, 18.2 +/- 1.2 ng/min.follicle; 10 ng/ml EGF, 11.9 +/- 0.8) by the perfused ovary. In contrast, EGF did not inhibit hCG-induced progesterone secretion by isolated granulosa cells. Ovulatory efficiency (number of ovulated ova per number of mature follicles x 100) when EGF was given 30 min before hCG was reduced dose-dependently from 58.2% with no EGF to 8.3% with 10 ng/ml EGF (P less than 0.001). Ovulation was not inhibited by EGF when it was given 30 min after hCG. Distribution of hCG in the preovulatory follicle was confined to the basement membrane, thecal cell layer, and a small fraction of the outer granulosa cell layer. These observations suggest that gonadotropin stimulates the follicle through the release of a secondary signal(s) from ligand-bound granulosa cells near the follicle wall to unexposed cells of the inner avascular area. EGF may inhibit the follicular response to hCG by attenuation of this cell to cell communication.

Evidence for the production of a nonsteroidal endotheliotropic factor by human granulosa cells in culture.

OBJECTIVE: To determine the cellular source of the angiogenic activity displayed by follicular fluid (FF). DESIGN: Human granulosa cells were harvested from 27 follicular aspirates obtained 34 to 36 hours after eight patients, previously treated with human menopausal gonadotropin (hMG), follicle-stimulating hormone plus hMG, or clomiphene citrate and hMG received human chorionic gonadotropin (10,000 IU intramuscularly). Granulosa cells from individual follicles were plated at 50,000 cells/cm2 in Medium 199 (Sigma, St. Louis, MO) supplemented with either 5% calf serum or 0.1% bovine serum albumin; media collected 24 hours later was assayed in vitro measuring endothelial cell migration. Fractions depleted of steroids by reversed phase C1 chromatography were assayed as well. RESULTS: Granulosa cell-conditioned media from 18 of 27 follicles significantly stimulated endothelial cell migration (P less than 0.05). Chemoattractant activity did not appear to be related to steroid accumulation in the media and was not diminished in steroid depleted fractions. CONCLUSIONS: These findings suggest that human granulosa cells are a source of (nonsteroidal) endotheliotropic-angiogenic activity in FF.

Euthyroid thyrotoxic periodic paralysis.

Thyrotoxic periodic paralysis (TPP) is a dramatic complication of thyrotoxicosis usually seen in young men with untreated Graves' disease. We report the case of a 29-year-old active duty man with TPP attacks atypical in that they occurred during and after resolution of the hyperthyroidism. Our literature review revealed only two previously reported cases of TPP concurrent with euthyroidism. Risk factors for TPP include the postprandial state after carbohydrate-rich meals and the post-exertional state. At least a 2-week "window of vulnerability" for TPP appears to exist after initiation of antithyroid therapy. Hyperthyroid active duty males are especially at risk of TPP, and require physical profiling at the time of diagnosis and for a limited period after they become euthyroid, to minimize the occurrence of this complication.

Preliminary characterization of angiogenic activity in media conditioned by cells from luteinized rat ovaries.

Angiogenic activity was detected in media conditioned by ovarian cells from superovulated, pseudopregnant (PMSG/human CG treated) immature Holtzman rats. Media conditioned by cells from luteinized rat ovaries stimulated the directed migration of rabbit endothelial cells or mouse Balb/c3T3 cells, but was not mitogenic to either cell type. That endotheliotropic activity was not associated with a steroid was indicated by the finding that chemoattractant activity was detected in fractions after reversed-phase C18 chromatography, which removes more than 95% of steroids present in the media, and that chemoattractant activity was precipitated by ammonium sulfate and by ethanol. Full chemoattractant activity was recovered after boiling (95 C for 30 min), lyophilization, dialysis, Sephadex G-25 desalting columns, and pH changes from 3-10. After Sephadex G-200 chromatography, chemoattractant activity emerged at elution volumes corresponding to 20,000-30,000 mol wt. Chemoattractant activity was not retained by Concanavalin A-Sepharose or gelatin-Sepharose, and was only partially retained by heparin-agarose. Chemoattractant activity was also partially retained on both cation and anion exchange columns. Our collective findings indicate the presence of a nonsteroidal, heat-stable, pronase-sensitive factor, nominal mol wt of 20,000-30,000, in media conditioned by cells from luteinized rat ovaries; this factor is chemoattractive but not mitogenic to endothelial cells. Ovarian-derived chemoattractant activity appears to be distinct from fibroblast growth factor because it lacked detectable mitogenic activity, and because fibroblast growth factor was not active in our cell migration bioassay. Because stimulation of endothelial cell migration is a key event during angiogenesis, demonstration of an ovarian endotheliotropic chemoattractant is consistent with our hypothesis that angiogenesis factors play a role in the paracrine regulation of ovarian function.

Severe systemic reactions to typhoid vaccination: two cases and a review of the literature.

Typhoid vaccination is part of the routine immunization of all military personnel on mobility status. We report the cases of Air Force students who presented to our facility with presumed systemic reactions to the typhoid vaccine much more severe than those commonly reported. Review of the literature reveals that an influenza-like reaction of variable severity commonly occurs in these patients, and in addition there are isolated reports of more severe reactions. These findings shed doubt on the overall safety of the currently used vaccine and warrant a consideration of its abandonment. Several safer alternatives, including the new live oral vaccine used in Europe, are discussed.

Thyroid angiogenesis: endotheliotropic chemoattractant activity from rat thyroid cells in culture.

Thyroid enlargement in response to chronic hypersecretion of TSH reflects the coordinated growth of both parenchyma and stroma. Because Wollman et al. observed in propylthiouracil-fed rats that enlargement and remodeling of thyroid capillaries were strictly localized around follicles, they hypothesized that growth of perifollicular blood vessels is stimulated by angiogenic factors secreted by neighboring follicular epithelial cells. In support of this hypothesis, we report that media conditioned by rat thyroid cells were very active in an in vitro angiogenesis bioassay that measures stimulation of endothelial cell migration through chemotaxis membranes in microwell Boyden chamber assemblies. Primary cultures of thyroid cells from collagenase-dispersed glands from male or female Holtzman rats fed 0.01% propylthiouracil in the drinking water released activity that produced up to 5-fold increases in endothelial cell migration rates relative to those in identical unconditioned medium. Thyroid-derived activity was primarily chemotactic (i.e. only weakly chemokinetic) to both rabbit aortic and microvascular endothelial cells. That endotheliotropic activity is derived from thyroid parenchyma is indicated by the finding that media conditioned by FRTL cells, a clonally derived thyroid follicular epithelial cell line, produced parallel chemoattractant responses. Thyroid-conditioned media were also chemoattractant to mouse BALB/c-3T3 cells, which have endothelial cell characteristics. In contrast, thyroid-conditioned media did not increase the high spontaneous migration rate of Walker rat sarcoma (WR256) cells. T4, T3, thyroglobulin, bovine fibroblast growth factor (alpha and beta), and media conditioned by rabbit endothelial cells were inactive. Chemoattractant activity in serum containing conditioned media was retained by both 10,000 and 30,000 mol wt cut-off (MWCO) ultrafilters. Activity in serum-free thyroid-conditioned media was largely retained by 10,000 MWCO filters, but only partially retained by 30,000 MWCO filters; activity in the 30,000 filtrate was recoverable in a 10,000 MWCO retentate. These findings support the hypothesis that capillary growth during thyroid enlargement occurs, at least in part, as a result of a parenchymal-stromal (epithelial-mesenchymal) paracrine interaction mediated by specific endotheliotropic (angiogenic) factors released by follicular epithelial cells and distinct from T3, T4, and thyroglobulin.