Stereotactic image-guided neoadjuvant ablative single-dose radiation, then lumpectomy, for early breast cancer: the SIGNAL prospective single-arm trial of single-dose radiation therapy.

Background and Purpose: Adjuvant whole-breast irradiation after breast-conserving surgery, typically delivered over several weeks, is the traditional standard of care for low-risk breast cancer. More recently, hypofractionated, partial-breast irradiation has increasingly become established. Neoadjuvant single-fraction radiotherapy (rt) is an uncommon approach wherein the unresected lesion is irradiated preoperatively in a single fraction. We developed the signal (Stereotactic Image-Guided Neoadjuvant Ablative Radiation Then Lumpectomy) trial, a prospective single-arm trial to test our hypothesis that, for low-risk carcinoma of the breast, the preoperative single-fraction approach would be feasible and safe. Methods: Patients presenting with early-stage (T < 3 cm), estrogen-positive, clinically node-negative invasive carcinoma of the breast with tumours at least 2 cm away from skin and chest wall were enrolled. All patients received prone breast magnetic resonance imaging (mri) and prone computed tomography simulation. Treatable patients received a single 21 Gy fraction of external-beam rt (as volumetric-modulated arc therapy) to the primary lesion in the breast, followed by definitive surgery 1 week later. The primary endpoints at 3 weeks, 6 months, and 1 year were toxicity and cosmesis (that is, safety) and feasibility (defined as the proportion of mri-appropriate patients receiving rt). Results: Of 52 patients accrued, 27 were successfully treated. The initial dosimetric constraints resulted in a feasibility failure, because only 57% of eligible patients were successfully treated. Revised dosimetric constraints were developed, after which 100% of patients meeting mri criteria were treated according to protocol. At 3 weeks, 6 months, and 1 year after the operation, toxicity, patient- and physician-rated cosmesis, and quality of life were not significantly different from baseline. Conclusions: The signal trial presents a feasible method of implementing single-dose preoperative rt in early-stage breast cancer. This pilot study did not identify any significant toxicity and demonstrated excellent cosmetic and quality-of-life outcomes. Future randomized multi-arm studies are required to corroborate these findings.

The significance of circulating tumor cells in prostate cancer patients undergoing adjuvant or salvage radiation therapy.

BACKGROUND: Following radical prostatectomy, success of adjuvant and salvage radiation therapy (RT) is dependent on the absence of micrometastatic disease. However, reliable prognostic/predictive factors for determining this are lacking. Therefore, novel biomarkers are needed to assist with clinical decision-making in this setting. Enumeration of circulating tumor cells (CTCs) using the regulatory-approved CellSearch System (CSS) is prognostic in metastatic prostate cancer. We hypothesize that CTCs may also be prognostic in the post-prostatectomy setting. METHODS: Patient blood samples (n=55) were processed on the CSS to enumerate CTCs at 0, 6, 12 and 24 months after completion of RT. CTC values were correlated with predictive/prognostic factors and progression-free survival. RESULTS: CTC status (presence/absence) correlated significantly with positive margins (increased likelihood of CTC(neg) disease; P=0.032), and trended toward significance with the presence of seminal vesicle invasion (CTC(pos); P=0.113) and extracapsular extension (CTC(neg); P=0.116). Although there was a trend toward a decreased time to biochemical failure (BCF) in baseline CTC-positive patients (n=9), this trend was not significant (hazard ratio (HR)=0.3505; P=0.166). However, CTC-positive status at any point (n=16) predicted for time to BCF (HR=0.2868; P=0.0437). CONCLUSIONS: One caveat of this study is the small sample size utilized (n=55) and the low number of patients with CTC-positive disease (n=16). However, our results suggest that CTCs may be indicative of disseminated disease and assessment of CTCs during RT may be helpful in clinical decision-making to determine, which patients may benefit from RT versus those who may benefit more from systemic treatments.

5-HT1B autoreceptors differentially modulate the expression of conditioned fear in a circuit-specific manner.

Located in the nerve terminals of serotonergic neurons, 5-HT1B autoreceptors are poised to modulate synaptic 5-HT levels with precise temporal and spatial control, and play an important role in various emotional behaviors. This study characterized two novel, complementary viral vector strategies to investigate the contribution of 5-HT1B autoreceptors to fear expression, displayed as freezing, during contextual fear conditioning. Increased expression of 5-HT1B autoreceptors throughout the brain significantly decreased fear expression in both wild-type (WT) and 5-HT1B knockout (1BKO) mice when receptor levels were increased with a cell-type-specific herpes simplex virus (HSV) vector injected into the dorsal raphe nucleus (DRN). Additional studies used an intersectional viral vector strategy, in which an adeno-associated virus containing a double-floxed inverted sequence for the 5-HT1B receptor (AAV-DIO-1B) was combined with the retrogradely transported canine adenovirus-2 expressing Cre (CAV-Cre) in order to increase 5-HT1B autoreceptor expression only in neurons projecting from the DRN to the amygdala. Surprisingly, selective expression of 5-HT1B autoreceptors in just this circuit led to an increase in fear expression in WT, but not 1BKO, mice. These results suggest that activation of 5-HT1B autoreceptors throughout the brain may have an overall effect of attenuating fear expression, but activation of subsets of 5-HT1B autoreceptors in particular brain regions, reflecting distinct projections of serotonergic neurons from the DRN, may have disparate contributions to the ultimate response.

mu Opioid receptor-mediated G-protein activation by heroin metabolites: evidence for greater efficacy of 6-monoacetylmorphine compared with morphine.

The efficacy of heroin metabolites for the stimulation of mu opioid receptor-mediated G-protein activation was investigated using agonist-stimulated [(35)S]guanosine-5'-O-(gamma-thio)-triphosphate binding. In rat thalamic membranes, heroin and its primary metabolite, 6-monoacetylmorphine (6-MAM), were more efficacious than morphine or morphine-6-beta D-glucuronide. This increased efficacy was not due to increased action of heroin and 6-MAM at delta receptors, as determined by competitive antagonism by naloxone, lack of antagonism by naltrindole, and competitive partial antagonism with morphine. In agreement with this interpretation, the same relative efficacy profile of heroin and its metabolites was observed at the cloned human mu opioid receptor expressed in C6 glioma cells. Moreover, these efficacy differences were GDP-dependent in a manner consistent with accepted mechanisms of receptor-mediated G-protein activation. The activity of heroin was attributed to in vitro deacetylation to 6-MAM, as confirmed by HPLC analysis. These results indicate that the heroin metabolite 6-MAM possesses higher efficacy than other heroin metabolites at mu opioid receptors, which may contribute to the higher efficacy of heroin compared with morphine in certain behavioral paradigms in vivo.

Localization of 5-HT(7) receptors in rat brain by immunocytochemistry, in situ hybridization, and agonist stimulated cFos expression.

5-HT(7) receptors are recently identified members of the serotonin receptor family that have moderate to high affinity for several important psychotropic drugs. However, the lack of selective ligands has impeded the study of the brain distribution of these receptors. In this report, we describe the localization of 5-HT(7) receptor in rat forebrain by immunocytochemistry, in situ hybridization of 5-HT(7) mRNA, and functional stimulation of cFOS expression by 5-HT(7) receptor activation. The anatomical localization of 5-HT(7) mRNA in situ hybridization signal. Prominent immunostaining was apparent in numerous sites within the cerebral cortex, hippocampal formation, tenia tecta, thalamus and hypothalamus. 5-HT(7) receptors were detected in suprachiasmatic nucleus by both immunocytochemistry and in situ hybridization. At a microscopic level, both cell bodies and proximal fibers were strongly stained in these regions, suggesting a somatodendritic subcellular distribution. 5-HT(7) receptor-like immunoreactivity was further compared with 5-HT(7) mediated biological function by administering 8-OH-DPAT intracerebroventricular injection (icv)with WAY 100135 (to block 5-HT(1A) receptors) followed by double immunostaining localization of cFos activation and 5-HT(7) receptors. In all regions examined, cFos stimulation and 5-HT(7)-like immunoreactivity colocalized to the same neurons. Furthermore, cFos activation by 8-OH-DPAT was blocked by pimozide--a 5-HT(7) antagonist. Therefore, by using multiple strategies, we were able to localize 5-HT(7) receptors in rat brain unequivocally. The distribution of these receptors is consistent with their involvement in the control of circadian activity and the action of anti-depressants and atypical neuroleptics.

A new model of the blood--brain barrier: co-culture of neuronal, endothelial and glial cells under dynamic conditions.

Developing in vitro blood-brain barrier (BBB) models that closely mimic the natural state is important for theoretical and practical applications, including drug development. We previously developed an in vitro BBB model based on co-culturing endothelial cells with glia in the presence of flow on hollow fiber tube culture substrates. We now report that this dynamic in vitro BBB (DIV-BBB) can be successfully used to co-culture differentiated serotonergic neurons in the presence of a BBB. These neurons demonstrated fluoxetine-sensitive serotonin (5HT) uptake and depolarization-induced release of [3H]5HT. Our results demonstrate that the DIV-BBB is a suitable model for culturing of neurons in a quasi-physiological microenvironment and in the presence of a high-resistance, stereoselective BBB.

Ketorolac versus fentanyl for postoperative pain management in outpatients.

OBJECTIVE: The purpose of this study was to compare the efficacy and safety of i.v. ketorolac and fentanyl for moderate to severe postoperative pain in patients undergoing elective surgery in an ambulatory surgery unit. DESIGN: A double-blind randomized trial. SETTING: An ambulatory surgery unit in a university-affiliated hospital. PATIENTS: Sixty-nine patients undergoing elective laparoscopy, inguinal hernia repair, or knee arthroscopy were enrolled. INTERVENTION: Patients were randomly assigned to receive intravenous ketorolac 30 mg (n = 38) or fentanyl 50 micrograms (n = 31) for moderate to severe postoperative pain. OUTCOME MEASURES: Pain, assessed using a 100-mm visual analog scale and a 5-point verbal pain scale; adverse effects, as well as vital signs were recorded every 15 min for 150 min or until discharge from the postanesthesia care unit, 6 and 24 h after discharge. RESULTS: Pain reduction on both visual analog and verbal scales was significantly greater with fentanyl than ketorolac at 15 min. In addition, the proportion of patients requiring remedication at the 15-min time point was significantly greater in the ketorolac group. However, there were no significant differences between fentanyl and ketorolac between 30 and 150 min after surgery. Notably, pain reduction was significantly greater with ketorolac on the verbal scale at the 6 h measurement. CONCLUSIONS: Ketorolac appears not be as effective as fentanyl in treating early postoperative pain. Although fentanyl still appears to be the drug of choice in the early postoperative period, the parenteral use of ketorolac was more effective during the later postoperative period in providing longer lasting analgesia.

Immunohistochemical detection of sex steroid receptors in breast cancer using routine paraffin sections: comparison with frozen sections and enzyme immunoassay.

Estrogen and progesterone receptors (ER and PR) on 48 surgically removed breast cancers were evaluated by three methods: immunoenzymatic (ER/PR-EIA), immunohistochemical in frozen sections (ER/PR-ICA), and immunohistochemical in paraffin sections (ER/PR-PAR). The monoclonal antibodies H222 and KD68 were used for immunohistochemical detection of ER and PR, respectively. Immunohistochemical stains for pS2, an estrogen-regulated protein, were also done for compatibility with previous ER/PR-PAR studies. We assessed concordance with chi-square and Pearson's correlation coefficient. We concluded that ER/PR-PAR is the least sensitive of the three assays (90.9% ER, 88.5% PR) and, until appropriate technical and clinical validation is achieved, should not be used as a first-line assay. Also, because of this lower sensitivity, we recommended that pS2 evaluation always accompany ER/PR-PAR to facilitate distinction between clinical negatives and those due to irretrievably lost immunoreactivity. We also conclude that ER/PR-ICA is a good semi-quantitative method that, in combination with ER/PR-EIA, most accurately assesses receptor status.

Mechanisms of deregulated her2/neu expression in breast-cancer cell-lines.

Mechanisms that result in HER2/neu over-expression in breast cancer were examined by studying breast cancer cell lines that express much higher levels of HER2/neu mRNA than normal breast tissue while maintaining a near normal HER2/neu gene copy number. Nuclear run-on experiments indicate that the breast cancer cell lines MDA-MB453, BT483 and BT474 have an increased HER2/neu gene transcription rate. By using HER2/neu promoter-CAT constructs, we show that the enhanced HER2/neu transcription rate in MDA-MB453 is due to activation of the gene in trans. We have localized a 13-bp element on the gene promoter that is required for the increased transcription rate and have demonstrated sequence specific interaction of this fragment with a nuclear protein complex. We have also shown that in BT474 cells, transcriptional upregulation is mainly due to gene amplification and does not fully account for the levels of HER2/neu mRNA, demonstrating that deregulation of HER2/neu expression at the post-transcriptional level significantly contributes to HER2/neu overexpression in BT474 cells. Our results suggest that multiple activations of the HER2/neu gene are involved in HER2/neu over-expression in breast cancer lines and that multiple mechanisms may function simultaneously within a single cell line.

Biochemical and genetic basis for the ETS (enterotoxin sensitivity) phenotype in mice.

Evidence has been accumulating that humans are genetically predisposed to cholera gravis. Using the sealed adult mouse model, we found that certain inbred mice were also hypersensitive to cholera toxin (CT). Such mice were designated ETS+ (enterotoxin sensitive), and the trait was linked to the K end of the mouse H-2 histocompatibility complex. Cells isolated from ETS+ mice bound more CT and accumulated more cyclic adenosine monophosphate (cAMP) after intoxication. Analysis of ETS+ cells showed that they express lesser amounts of the non-GM1 gangliosides that block or compete for relevant CT binding sites in ETS- cells. Conversion of ETS- non-GM1 gangliosides to GM1 with neuraminidase increased CT binding and cAMP responses. Reconstitution of nonreactive ganglioside-deficient cells with ETS+ or ETS- gangliosides caused them to bind CT like the original ETS+ or ETS- cells. Ganglioside expression genes known to map to the same H-2-linked region as the ETS phenotype seem to be involved in controlling murine susceptibility to CT.

Opioid and cannabinoid receptor inhibition of adenylyl cyclase in brain.

Both opioids and cannabinoids bind to G-protein-coupled receptors to inhibit adenylyl cyclase in neurons. These reactions were assayed in brain membranes, where maximal inhibitory activity occurred in the following regions: mu-opioid inhibition in rat thalamus, delta-opioid inhibition in rat striatum, kappa-opioid inhibition in guinea pig cerebellum, and cannabinoid inhibition in cerebellum. The inhibition of adenylyl cyclase by both cannabinoid and opioid agonists was typical of G-protein-linked receptors: they required GTP, they were not supported by non-hydrolyzable GTP analogs, and they were abolished (in primary neuronal cell culture) by pertussis toxin treatment. The immediate targets of this system were determined by assaying protein phosphorylation in the presence of receptor agonists and App(NH)p, a substrate for adenylyl cyclase. In striatal membranes, opioid agonists inhibited the phosphorylation of at least two bands of MW 85 and 63 kDa, which may be synapsins I and II, respectively. Other experiments determined the long-term effects of this second messenger system. In primary neuronal cultures, opioid-inhibited adenylyl cyclase attenuated forskolin-stimulated pro-enkephalin mRNA levels, thus providing a feedback regulation of opioid synthesis. Finally, in cerebellar granule cells, both cannabinoid and opioid receptors may exist on the same cells. In these cells, agonists which bind to different receptor types may produce similar biological responses.

Light-dependent changes in psbD and psbC transcripts of barley chloroplasts: accumulation of two transcripts maintains psbD and psbC translation capability in mature chloroplasts.

The psbD and psbC genes encode two polypeptides of Photosystem II. These genes are adjacent in the barley chloroplast genome and are part of a 5.7 kbp transcription unit. In dark-grown barley, four large transcripts hybridize to psbD and psbC; two additional transcripts hybridize to psbC. Illumination of 4.5-day-old dark-grown seedlings causes a decrease in the six psbD--psbC transcripts found in etioplasts and the accumulation of two different transcripts of 4.0 and 3.2 kb which hybridize to psbD and psbC. The light-induced transcripts have a common 5' end approximately 600 nt upstream of psbD and 3' ends 1175 and 175 nt downstream of psbC. The shift in psbD--psbC transcript population occurs during a phase of chloroplast maturation when transcript levels and translation of chloroplast genes such as psaA--psaB and psbB decline approximately 3- to 5-fold. In contrast, translation of the psbD and psbC gene products declines to a lesser extent, suggesting that the light-induced accumulation of the 4.0 and 3.2 kb psbD--psbC transcripts is required to maintain psbD and psbC gene product translation in mature chloroplasts.

Comparison of various cryoprotective agents on washed chicken spermatozoa. 5. Effect of glucose, sucrose and polyvinylpyrrolidone.

A study was conducted to determine the effect of glucose, sucrose and polyvinylpyrrolidone (PVP) on the viability of chicken spermatozoa. Levels of 4, 8 and 12% glucose, sucrose or 12% PVP significantly reduced the motility and fertilizing capacity of spermatozoa. Adding the compounds in bulk or fractions eliminated the drastic effect of 4% sucrose on motility and fertility but proved ineffective with 4% glucose or 12% PVP. A complete loss of motility with no effect on fertility was observed with 6% PVP was combined with either ethylene glycol or DMSO.