Identification of galanin and its receptor GalR1 as novel determinants of resistance to chemotherapy and potential biomarkers in colorectal cancer.

PURPOSE: A major factor limiting the effective clinical management of colorectal cancer (CRC) is resistance to chemotherapy. Therefore, the identification of novel, therapeutically targetable mediators of resistance is vital. EXPERIMENTAL DESIGN: We used a CRC disease-focused microarray platform to transcriptionally profile chemotherapy-responsive and nonresponsive pretreatment metastatic CRC liver biopsies and in vitro samples, both sensitive and resistant to clinically relevant chemotherapeutic drugs (5-FU and oxaliplatin). Pathway and gene set enrichment analyses identified candidate genes within key pathways mediating drug resistance. Functional RNAi screening identified regulators of drug resistance. RESULTS: Mitogen-activated protein kinase signaling, focal adhesion, cell cycle, insulin signaling, and apoptosis were identified as key pathways involved in mediating drug resistance. The G-protein-coupled receptor galanin receptor 1 (GalR1) was identified as a novel regulator of drug resistance. Notably, silencing either GalR1 or its ligand galanin induced apoptosis in drug-sensitive and resistant cell lines and synergistically enhanced the effects of chemotherapy. Mechanistically, GalR1/galanin silencing resulted in downregulation of the endogenous caspase-8 inhibitor FLIP(L), resulting in induction of caspase-8-dependent apoptosis. Galanin mRNA was found to be overexpressed in colorectal tumors, and importantly, high galanin expression correlated with poor disease-free survival of patients with early-stage CRC. CONCLUSION: This study shows the power of systems biology approaches to identify key pathways and genes that are functionally involved in mediating chemotherapy resistance. Moreover, we have identified a novel role for the GalR1/galanin receptor-ligand axis in chemoresistance, providing evidence to support its further evaluation as a potential therapeutic target and biomarker in CRC.

Calbindin 2 (CALB2) regulates 5-fluorouracil sensitivity in colorectal cancer by modulating the intrinsic apoptotic pathway.

The role of the calcium binding protein, Calbindin 2 (CALB2), in regulating the response of colorectal cancer (CRC) cells to 5-Fluorouracil (5-FU) was investigated. Real-time RT-PCR and Western blot analysis revealed that CALB2 mRNA and protein expression were down-regulated in p53 wild-type and p53 null isogenic HCT116 CRC cell lines following 48 h and 72 h 5-FU treatment. Moreover, 5-FU-induced apoptosis was significantly reduced in HCT116 and LS174T CRC cell lines in which CALB2 expression had been silenced. Further investigation revealed that CALB2 translocated to the mitochondria following 5-FU treatment and that 5-FU-induced loss of mitochondrial membrane potential (Δψ(m)) was abrogated in CALB2-silenced cells. Furthermore, CALB2 silencing decreased 5-FU-induced cytochrome c and smac release from the mitochondria and also decreased 5-FU-induced activation of caspases 9 and 3/7. Of note, co-silencing of XIAP overcame 5-FU resistance in CALB2-silenced cells. Collectively, these results suggest that following 5-FU treatment in CRC cell lines, CALB2 is involved in apoptosis induction through the intrinsic mitochondrial pathway. This indicates that CALB2 may be an important mediator of 5-FU-induced cell death. Moreover, down-regulation of CALB2 in response to 5-FU may represent an intrinsic mechanism of resistance to this anti-cancer drug.

Atlanto-occipital dislocation with retroclival hematoma in a pediatric patient presenting to the emergency department.

Atlanto-occipital dislocation is a rare, oftentimes fatal injury sustained from high-impact trauma. It is seen more often in children compared with adults. In the past decade, there are more pediatric survivors presenting to the emergency department for treatment. This case reviews the presentation, diagnosis, and treatment of a child who survived this injury.

Cardiac index measurements by transcutaneous Doppler ultrasound and transthoracic echocardiography in adult and pediatric emergency patients.

INTRODUCTION: Non-invasive hemodynamic monitoring may facilitate resuscitation in critically ill patients. Validation studies examining a transcutaneous Doppler ultrasound technology, USCOM-1A, using pulmonary artery catheter as the reference standard showed varying results. In this study, we compared non-invasive cardiac index (CI) measurements by USCOM-1A with transthoracic echocardiography (TTE). METHODS: This study was a prospective, observational cohort study at a university tertiary-care emergency department, enrolling a convenience sample of adult and pediatric patients. Paired measures of CI, stroke volume index (SVI), aortic outflow tract diameter (OTD), velocity time integral (VTI) were obtained using USCOM-1A and TTE. Pearson's correlation and Bland-Altman analyses were performed. RESULTS: One-hundred and sixteen subjects were enrolled, with obtainable USCOM-1A CI measurements for 99 subjects (55 adults age 50 +/- 20 years and 44 children age 11 +/- 4 years) in the final analysis. Cardiac, gastrointestinal and infectious illnesses were the most common presenting diagnostic categories. The reference standard TTE measurements of CI, SVI, OTD, and VTI in all subjects were 3.08 +/- 1.18 L/min/m(2), 37.10 +/- 10.91 mL/m(2), 1.92 +/- 0.36 cm, and 20.36 +/- 4.53 cm, respectively. Intra-operator reliability of USCOM-1A CI measurements showed a correlation coefficient of r = 0.79, with 11 +/- 22% difference between repeated measures. The bias and limits of agreement of USCOM-1A compared to TTE CI were 0.58 (-1.48 to 2.63) L/min/m(2). The percent difference in CI measurements with USCOM-1A was 31 +/- 28% relative to TTE measurements. CONCLUSIONS: The USCOM-1A hemodynamic monitoring technology showed poor correlation and agreement to standard transthoracic echocardiography measures of cardiac function. The utility of USCOM-1A in the management of critically ill patients remains to be determined.

Myasthenia gravis in an adolescent patient presenting to the pediatric emergency department.

Myasthenia gravis is a rare, chronic, autoimmune disorder characterized by postsynaptic dysfunction at the neuromuscular junction. The disease affects more females than males. We describe the case of a 17-year-old female adolescent with recurrent episodes of dysarthria and dysphagia and a history of aspiration pneumonia. A bedside edrophonium (Tensilon) test in our emergency department confirmed the diagnosis of myasthenia gravis.

BRCA1 transcriptionally regulates genes associated with the basal-like phenotype in breast cancer.

Expression profiling of BRCA1-deficient tumours has identified a pattern of gene expression similar to basal-like breast tumours. In this study, we examine whether a BRCA1-dependent transcriptional mechanism may underpin the link between BRCA1 and basal-like phenotype. In methods section, the mRNA and protein were harvested from a number of BRCA1 mutant and wild-type breast cancer cell lines and from matched isogenic controls. Microarray-based expression profiling was used to identify potential BRCA1-regulated transcripts. These gene targets were then validated (by in silico analysis of tumour samples) by real-time PCR and Western blot analysis. Chromatin immunoprecipitation (ChIP) assays were used to confirm recruitment of BRCA1 to specific promoters. In results, we demonstrate that functional BRCA1 represses the expression of cytokeratins 5(KRT5) and 17(KRT17) and p-Cadherin (CDH3) in HCC1937 and T47D breast cancer cell lines at both mRNA and protein level. ChIP assays demonstrate that BRCA1 is recruited to the promoters of KRT5, KRT17 and CDH3, and re-ChIP assays confirm that BRCA1 is recruited independently to form c-Myc and Sp1 complexes on the CDH3 promoter. We show that siRNA-mediated inhibition of endogenous c-Myc (and not Sp1) results in a marked increase in CDH3 expression analogous to that observed following the inhibition of endogenous BRCA1. The data provided suggest a model whereby BRCA1 and c-Myc form a repressor complex on the promoters of specific basal genes and represent a potential mechanism to explain the observed overexpression of key basal markers in BRCA1-deficient tumours.

A work sampling study of provider activities in school-based health centers.

BACKGROUND: The purpose of this study was to describe provider activities in a convenience sample of School-Based Health Centers (SBHCs). The goal was to determine the relative proportion of time that clinic staff engaged in various patient care and non-patient care activities. METHODS: All provider staff at 4 urban SBHCs participated in this study; 2 were in elementary schools, 1 in a middle school, and 1 in a school with kindergarten through grade 8. The study examined provider activity from 6 days sampled at random from the school year. Participants were asked to document their activities in 15-minute intervals from 8:00 a.m. to 5:00 p.m. A structured recording form was used that included 35 activity categories. RESULTS: Overall, 1492 records were completed, accounting for 2708 coded activities. Almost half (48%) of all staff activities were coded as direct patient contact, with clinic operations the second largest category. Limited variations in activities were found across clinic sites and according to season. CONCLUSIONS: A significant amount of provider activity was directed at the delivery of health care; direct patient care and clinic operations combined accounted for approximately 75% of clinic activity. Patient, classroom, and group education activities, as well as contacts with parents and school staff accounted for 20% of all clinic activity and represent important SBHC functions that other productivity measures such as billing data might not consistently track. Overall, the method was acceptable to professional staff as a means of tracking activity and was adaptable to meet their needs.

Inter-rater reliability for noninvasive measurement of cardiac function in children.

INTRODUCTION: A transcutaneous ultrasound monitor has recently been developed which noninvasively and quickly measures cardiac output. Validity and reliability testing has been reported in adults. No reliability testing has been undertaken in the pediatric population. OBJECTIVE: Our objective was to evaluate the inter-rater reliability of a transcutaneous Doppler ultrasound technique to measure cardiac index (CI) and stroke volume index (SVI) in pediatric emergency department patients. METHODS: An 8-month prospective observational study was conducted on a convenience sample of emergency department patients younger than 18 years old. Five raters were trained to use an ultrasound cardiac output monitoring device. Two raters, blinded to each other's results, obtained independent measurements from the same patient within 15 minutes of each other. Inter-rater agreement was measured with the Pearson product correlation coefficient. Bland-Altman analysis demonstrated the extent of deviation from a line of agreement between raters. RESULTS: Ninety-seven patients were enrolled. Major diagnostic categories included infection, trauma, and gastrointestinal disorders. There was significant inter-rater correlation for CI (r = 0.76; 95% confidence interval, 0.66Y0.83; P G 0.0001) and SVI (r = 0.79; 95% confidence interval, 0.70Y0.86; P G 0.0001). Bland-Altman analysis of CI measurements between 2 raters showed bias of 0.06, SD of bias 1.00, and 95% limits of agreement j1.91 to 2.02 L/min/m2. Stroke volume index showed bias of j0.5, SD of bias 11.01, and 95% limits of agreement j22.08 to 21.08 mL/m2. CONCLUSIONS: Transcutaneous Doppler ultrasound technique demonstrates acceptable inter-rater agreement for measuring CI and SVI in children.

Adjunctive atropine is unnecessary during ketamine sedation in children.

BACKGROUND: The prophylactic coadministration of atropine or other anticholinergics during dissociative sedation has historically been considered mandatory to mitigate ketamine-associated hypersalivation. Emergency physicians (EPs) are known to omit this adjunct, so a prospective study to describe the safety profile of this practice was initiated. OBJECTIVES: To quantify the magnitude of excessive salivation, describe interventions for hypersalivation, and describe any associated airway complications. METHODS: In this prospective observational study of emergency department (ED) pediatric patients receiving dissociative sedation, treating physicians rated excessive salivation on a 100-mm visual analog scale and recorded the frequency and nature of airway complications and interventions for hypersalivation. RESULTS: Of 1,090 ketamine sedations during the 3-year study period, 947 (86.9%) were performed without adjunctive atropine. Treating physicians assigned the majority (92%) of these subjects salivation visual analog scale ratings of 0 mm, i.e., "none," and only 1.3% of ratings were >or= 50 mm. Transient airway complications occurred in 3.2%, with just one (brief desaturation) felt related to hypersalivation (incidence 0.11%, 95% confidence interval = 0.003% to 0.59%). Interventions for hypersalivation (most commonly suctioning) occurred in 4.2%, with no occurrences of assisted ventilation or intubation. CONCLUSIONS: When adjunctive atropine is omitted during ketamine sedation in children, excessive salivation is uncommon, and associated airway complications are rare. Anticholinergic prophylaxis is not routinely necessary in this setting.

BRCA1 mRNA expression levels predict for overall survival in ovarian cancer after chemotherapy.

PURPOSE: We investigated whether BRCA1 mRNA expression levels may represent a biomarker of survival in sporadic epithelial ovarian cancer following chemotherapy treatment. EXPERIMENTAL DESIGN: The effect of loss of BRCA1 expression on chemotherapy response in ovarian cancer was measured in vitro using dose inhibition assays and Annexin V flow cytometry. Univariate and multivariate analyses were done to evaluate the relationship between BRCA1 mRNA expression levels and survival after chemotherapy treatment in 70 fresh frozen ovarian tumors. RESULTS: We show that inhibition of endogenous BRCA1 expression in ovarian cancer cell lines results in increased sensitivity to platinum therapy and decreased sensitivity to antimicrotubule agents. In addition, we show that patients with low/intermediate levels of BRCA1 mRNA have a significantly improved overall survival following treatment with platinum-based chemotherapy in comparison with patients with high levels of BRCA1 mRNA (57.2 versus 18.2 months; P = 0.0017; hazard ratio, 2.9). Furthermore, overall median survival for higher-BRCA1-expressing patients was found to increase following taxane-containing chemotherapy (23.0 versus 18.2 months; P = 0.12; hazard ratio, 0.53). CONCLUSIONS: We provide evidence to support a role for BRCA1 mRNA expression as a predictive marker of survival in sporadic epithelial ovarian cancer.

Molecular basis for estrogen receptor alpha deficiency in BRCA1-linked breast cancer.

BACKGROUND: BRCA1-mutant breast tumors are typically estrogen receptor alpha (ER alpha) negative, whereas most sporadic tumors express wild-type BRCA1 and are ER alpha positive. We examined a possible mechanism for the observed ER alpha-negative phenotype of BRCA1-mutant tumors. METHODS: We used a breast cancer disease-specific microarray to identify transcripts that were differentially expressed between paraffin-embedded samples of 17 BRCA1-mutant and 14 sporadic breast tumors. We measured the mRNA levels of estrogen receptor 1 (ESR1) (the gene encoding ER alpha), which was differentially expressed in the tumor samples, by quantitative polymerase chain reaction. Regulation of ESR1 mRNA and ER alpha protein expression was assessed in human breast cancer HCC1937 cells that were stably reconstituted with wild-type BRCA1 expression construct and in human breast cancer T47D and MCF-7 cells transiently transfected with BRCA1-specific short-interfering RNA (siRNA). Chromatin immunoprecipitation assays were performed to determine if BRCA1 binds the ESR1 promoter and to identify other interacting proteins. Sensitivity to the antiestrogen drug fulvestrant was examined in T47D and MCF-7 cells transfected with BRCA1-specific siRNA. All statistical tests were two-sided. RESULTS: Mean ESR1 gene expression was 5.4-fold lower in BRCA1-mutant tumors than in sporadic tumors (95% confidence interval [CI] = 2.6-fold to 40.1-fold, P = .0019). The transcription factor Oct-1 recruited BRCA1 to the ESR1 promoter, and both BRCA1 and Oct-1 were required for ER alpha expression. BRCA1-depleted breast cancer cells expressing exogenous ER alpha were more sensitive to fulvestrant than BRCA1-depleted cells transfected with empty vector (T47D cells, the mean concentration of fulvestrant that inhibited the growth of 40% of the cells [IC40] for empty vector versus ER alpha: >10(-5) versus 8.0 x 10(-9) M [95% CI = 3.1 x 10(-10) to 3.2 x 10(-6) M]; MCF-7 cells, mean IC40 for empty vector versus ER alpha: >10(-5) versus 4.9 x 10(-8) M [95% CI = 2.0 x 10(-9) to 3.9 x 10(-6) M]). CONCLUSIONS: BRCA1 alters the response of breast cancer cells to antiestrogen therapy by directly modulating ER alpha expression.

Test characteristics of parent's visual analog scale score in predicting ventriculoperitoneal shunt malfunction in the pediatric emergency department.

OBJECTIVES: Many parents of children with ventriculoperitoneal shunts present to the emergency department for evaluation of a possible shunt malfunction. No study to date has evaluated their ability to predict a shunt malfunction. Our study objective was to evaluate parents' accuracy for predicting a shunt malfunction in their child. We hypothesize that parents more experienced with prior shunt malfunctions are better able to predict subsequent malfunctions in their child. METHODS: We conducted a prospective, descriptive study on children younger than 18 years presenting to our tertiary care pediatric emergency department with a possible ventriculoperitoneal shunt malfunction. Parents rated the likelihood of a shunt malfunction using an unmarked 100-mm visual analog scale marked definitely malfunctioning at the high end. An experienced parent was defined as one who had previously experienced at least 3 shunt malfunctions in their child. RESULTS: We enrolled 85 parent-child dyads in our study. Twenty-four children were diagnosed with a malfunction. The predictive ability of parents to determine a shunt malfunction was found at a threshold visual analog scale score of 66 (sensitivity, 88.9%, and specificity, 62.2%). At a determined threshold value of 85 or more, experienced parents had a high specificity of 89.2% with a positive likelihood ratio of 5.1. Experienced parents showed an area under the curve of 0.7928 (95% confidence interval, 0.6037-0.9819); and inexperienced parents, 0.5611 (95% confidence interval, 0.3646-0.7576) (P = 0.096). CONCLUSIONS: Experienced parents are better able to predict a shunt malfunction in their child.

Signs and symptoms of cerebrospinal fluid shunt malfunction in the pediatric emergency department.

OBJECTIVES: Pediatric patients with cerebrospinal fluid shunts frequently present to the emergency department for evaluation of possible shunt malfunction. Most shunt studies appear in the neurosurgical literature. To our knowledge, none have reviewed presenting signs and symptoms of shunt malfunction in patients who present to the pediatric emergency department. The study objective was to evaluate the medical record of children with cerebrospinal fluid shunts who presented to a pediatric emergency department to determine if any signs and/or symptoms were predictive of shunt malfunction. METHODS: A retrospective chart review was conducted on 352 pediatric patients aged 0 to 18 years, who presented to the pediatric emergency department between January 1, 1998, and December 31, 2002, with signs and/or symptoms that prompted an evaluation for possible shunt malfunction. RESULTS: Univariate analysis of all signs and symptoms revealed lethargy (odds ratio, 1.99; 95% confidence interval, 1.15-3.42; P = 0.02) and shunt site swelling (odds ratio, 2.56, 95% confidence interval, 1.08-6.07, P = 0.03) to be significantly predictive of shunt malfunction. Logistic regression analysis continued to show significance for lethargy (odds ratio, 2.20; bias-corrected 95% confidence interval, 1.11-3.63) and shunt site swelling (odds ratio, 3.10; bias-corrected 95% confidence interval, 1.38-9.05), but found no other study variable to be significant. Bootstrap resampling validated the importance of the significant variables identified in the regression analysis. CONCLUSIONS: In this study, lethargy and shunt site swelling were predictive of shunt malfunction. Other signs and symptoms studied did not reach statistical significance; however, one must maintain a high index of suspicion when evaluating children with an intracranial shunt because the presentation of malfunction is widely varied. A missed diagnosis can result in permanent neurological sequelae or even death.

Albuterol nebulized in heliox in the initial ED treatment of pediatric asthma: a blinded, randomized controlled trial.

OBJECTIVE: A prospective blinded, randomized controlled trial was undertaken to compare the initial response of albuterol nebulized in heliox or control in the treatment of moderately severe asthma in children presenting to a pediatric ED. METHODS: Patients were randomized to receive heliox (n = 20) or control (n = 21). The primary outcome was to compare a modified dyspnea index score at 10 and 20 minutes after randomization. Secondary outcomes were to determine if heliox decreased admission rates or endotracheal intubation. RESULTS: There was no statistically significant difference found at 10 or 20 minutes after randomization with heliox (P = .169 and P = .062, respectively). No statistical difference in admission rate was found, and no patients required endotracheal intubation in either group. CONCLUSIONS: Our results demonstrate that albuterol nebulized with heliox offered no clinical benefit over standard therapy in the initial treatment of moderately severe asthma in the ED.

Perichondritis--a complication of high ear piercing.

Body piercing has become a fashionable trend in western culture. High ear piercing is common among the adolescent population and complications from this procedure although uncommon can be permanently disfiguring. We describe a case of perichondritis after high ear piercing in an adolescent male. A review of the history of body piercing, complications, risk factors, and treatment is presented.

BRCA1 and c-Myc associate to transcriptionally repress psoriasin, a DNA damage-inducible gene.

Evidence is accumulating to suggest that some of the diverse functions associated with BRCA1 may relate to its ability to transcriptionally regulate key downstream target genes. Here, we identify S100A7 (psoriasin), S100A8, and S100A9, members of the S100A family of calcium-binding proteins, as novel BRCA1-repressed targets. We show that functional BRCA1 is required for repression of these family members and that a BRCA1 disease-associated mutation abrogates BRCA1-mediated repression of psoriasin. Furthermore, we show that BRCA1 and c-Myc form a complex on the psoriasin promoter and that BRCA1-mediated repression of psoriasin is dependent on functional c-Myc. Finally, we show that psoriasin expression is induced by the topoisomerase IIalpha poison, etoposide, in the absence of functional BRCA1 and increased psoriasin expression enhances cellular sensitivity to this chemotherapeutic agent. Therefore, we identified a novel transcriptional mechanism that is likely to contribute to BRCA1-mediated resistance to etoposide.