Rapid Cycle Deliberate Practice Improves Retention of Pediatric Resuscitation Skills Compared With Postsimulation Debriefing.

INTRODUCTION: Rapid cycle deliberate practice (RCDP) for teaching team-based resuscitation is associated with similar improvements in immediate performance as compared with postsimulation debriefing (PSD). Limited studies compare skill retention between these 2 modalities. Our objective was to compare retention of team leader performance in residents trained with RCDP versus PSD. METHODS: This was a cluster-randomized trial comparing RCDP and PSD from January 2018 to April 2019. Pediatric and emergency medicine residents participated in simulation-based pediatric resuscitation education, and teams were randomized to undergo either RCDP or PSD. Each participant's team leader performance was assessed 1 to 12 months after training via a simulated cardiac arrest. The primary outcome was time to defibrillation. Secondary outcomes included overall team leader performance and time to chest compressions. RESULTS: Thirty-two residents (90.6% pediatrics, 9.4% emergency medicine) met inclusion criteria (16 RCDP, 16 PSD). Of the 32 residents, 40% returned in 1 to 3 months, 25% 3 to 6 months, 16% 6 to 9 months, and 19% 10 to 12 months. Participants in RCDP had more than 5 times the odds of achieving defibrillation versus those in the PSD group (odds ratio = 5.57, 95% confidence interval = 1.13-27.52, P = 0.04). The RCDP group had a higher mean Resident Team Leader Evaluation score (0.54 ± 0.19) than the PSD group (0.34 ± 0.16, P < 0.001). CONCLUSIONS: This study shows significant differences in subsequent performance in the team leader trained with RCDP and suggests that RCDP may improve retention of pediatric resuscitation skills compared with PSD. Future studies should focus on best applications for RCDP with attention to knowledge and skill decay.

Evolution of clinical event debriefs in a quaternary pediatric emergency department after implementation of a debriefing tool.

BACKGROUND: Debriefing clinical events in the emergency department (ED) can enhance team performance and provide mutual support. However, ED debriefing remains infrequent and nonstandardized. A clinical tool (DISCERN-Debriefing In Situ Conversation after Emergent Resuscitation Now) was developed to facilitate ED debriefing. To date, there are no studies providing qualitative analysis of clinical event debriefs done using such a tool. Our goal was to explore common themes elicited by debriefing following implementation of DISCERN. METHODS: This was a retrospective mixed-methods study analyzing DISCERN data from 2012 through 2017 in a pediatric ED. Quantitative data were analyzed using descriptive statistics. With constant comparison analysis, themes were categorized when applicable within the context of crisis resource management (CRM) principles, previously used as a framework for description of nontechnical skills. Member checking was performed to ensure trustworthiness. RESULTS: We reviewed 400 DISCERN forms. Overall, 170 (41.6%) of target clinical events were debriefed during the study period. The number of clinical events debriefed per year decreased significantly over the study period, from 118 debriefed events in 2013 to 20 debriefed events in 2017 (p < 0.001). Events were more likely to be debriefed if cardiopulmonary resuscitation was needed (odds ratio [OR] = 11.8, 95% confidence interval [CI] = 4.1-33.8]) or if the patient expired (OR = 8.9, 95% CI = 2.7-29.1]). CRM principles accounted for 81% of debriefing statements, focusing on teamwork, communication, and preparation, and these themes remained consistent throughout the study period. CONCLUSIONS: Use of the DISCERN tool declined over the study period. The DISCERN tool was utilized more commonly after the highest-acuity events. Clinical event debriefs aligned with CRM principles, with medical knowledge discussed less frequently, and the content of debriefs remained stable over time.

Development and implementation of an end-of-shift clinical debriefing method for emergency departments during COVID-19.

BACKGROUND: Multiple guidelines recommend debriefing after clinical events in the emergency department (ED) to improve performance, but their implementation has been limited. We aimed to start a clinical debriefing program to identify opportunities to address teamwork and patient safety during the COVID-19 pandemic. METHODS: We reviewed existing literature on best-practice guidelines to answer key clinical debriefing program design questions. An end-of-shift huddle format for the debriefs allowed multiple cases of suspected or confirmed COVID-19 illness to be discussed in the same session, promoting situational awareness and team learning. A novel ED-based clinical debriefing tool was implemented and titled Debriefing In Situ COVID-19 to Encourage Reflection and Plus-Delta in Healthcare After Shifts End (DISCOVER-PHASE). A facilitator experienced in simulation debriefings would facilitate a short (10-25 min) discussion of the relevant cases by following a scripted series of stages for debriefing. Data on the number of debriefing opportunities, frequency of utilization of debriefing, debriefing location, and professional background of the facilitator were analyzed. RESULTS: During the study period, the ED treated 3386 suspected or confirmed COVID-19 cases, with 11 deaths and 77 ICU admissions. Of the 187 debriefing opportunities in the first 8-week period, 163 (87.2%) were performed. Of the 24 debriefings not performed, 21 (87.5%) of these were during the four first weeks (21/24; 87.5%). Clinical debriefings had a median duration of 10 min (IQR 7-13). They were mostly facilitated by a nurse (85.9%) and mainly performed remotely (89.8%). CONCLUSION: Debriefing with DISCOVER-PHASE during the COVID-19 pandemic were performed often, were relatively brief, and were most often led remotely by a nurse facilitator. Future research should describe the clinical and organizational impact of this DISCOVER-PHASE.

Interpretation difficulty of normal versus abnormal radiographs using a pediatric example.

BACKGROUND: Radiograph teaching files are usually dominated by abnormal cases, implying that normal radiographs are easier to interpret. Our main objective was to compare the interpretation difficulty of normal versus abnormal radiographs of a set of common pediatric radiographs. METHODS: We developed a 234-item digital case bank of pediatric ankle radiographs, recruited a convenience sample of participants, and presented the cases to each participant who then classified the cases as normal or abnormal. We determined and contrasted the interpretation difficulty of the normal and abnormal x-rays items using Rasch Measurement Theory. We also identified case features that were associated with item difficulty. RESULTS: 139 participants (86 medical students, 7 residents, 29 fellows, 5 emergency physicians, and 3 radiologists) rated a minimum of 50 cases each, which resulted in 16,535 total ratings. Abnormal cases were more difficult (+0.99 logits) than were normal ones (-0.58 logits), difference 1.57 logits (95% CI 1.2, 2.0), but there was considerable overlap in difficulty scores. Patient variables associated with a more difficult normal radiograph included younger patient age (ß = -0.16, 95% CI -0.22, -0.10), history of distal fibular tenderness (ß = 0.55, 95% CI 0.17, 0.93), and presence of a secondary ossification centre (ß = 0.84, 95% CI 0.27, 1.41). CONCLUSIONS: While abnormal images were more difficult to interpret, normal images did show a range of interpretation difficulties. Including a significant proportion of normal cases may be of benefit to learners.

Effect of graded water immersion on vital capacity and plasma volume in patients with cervical spinal cord injury.

STUDY DESIGN: A case-control study was conducted. OBJECTIVE: The aim of the study was to test the hypothesis that the increase in hydrostatic pressure on the abdominal wall may be the major determinant of vital capacity (VC) improvement in tetraplegic subjects during water immersion, despite the blood volume shifts from the legs and abdomen to the thorax. SETTING: The study was carried out in the Rehabilitation Center, Brasília/DF, Brazil. METHODS: In total, 11 men with tetraplegia (complete motor lesion, C4-7, 30.4 years old) and 12 healthy controls were studied. Hematocrit level and spirometry values were obtained on dry land and at each level of immersion (the pelvis, xiphoid and neck). RESULTS: Baseline spirometry value of tetraplegic subjects showed reduced VC (53.3+/-17.4% of predicted), whereas all control subjects had >80% of predicted values. Neither group showed significant changes in VC at the pelvic and xiphoid levels of immersion. In tetraplegic subjects, VC increased by 27.2% at the neck level (+/-25.8, P<0.008), whereas in healthy subjects it decreased by 6.3% (+/-5.0, P<0.008). Both groups showed significantly increased inspiratory capacity only when immersed to the neck. Hematocrit level of tetraplegic subjects fell significantly with immersion to the xiphoid and neck levels (P<0.017), which occurred in controls only at the xiphoid level (P<0.017). CONCLUSIONS: Hydrostatic compression may be the main contributor to improving VC in tetraplegic subjects immersed in water. This improvement occurs despite increased plasma volume during immersion.

Maximal static mouth respiratory pressure in spinal cord injured patients: correlation with motor level.

STUDY DESIGN: Transversal. OBJECTIVES: The few studies concerning maximal static mouth respiratory pressures in patients with spinal cord lesions suggest a marked reduction. We studied the correlation of these parameters with the motor level of injury. SETTING: Rehabilitation Center, Brasília/DF, Brazil. METHODS: One hundred and thirty-one patients with traumatic spinal cord injury (C4-L3) were recruited. The participants were assessed by standard spirometry and maximal static mouth respiratory pressure. RESULTS: Forced vital capacity was most reduced in tetraplegics (subgroup C4-C5, 49%+/-25 predicted) and increase successively for each descending subgroup (C6-C8, 61%+/-22 predicted; T1-T6, 70%+/-15 predicted), becoming normal in low paraplegia (T7-L3, 84%+/-15 predicted). There is no evidence of an obstructive disturbance throughout all groups. The lowest average percent predicted of maximal static inspiratory pressure (MIP) was in the subgroup C4-C5 (50%+/-23). The average percent predicted of maximal static expiratory pressure (MEP) improved from 19%+/-14 in the C4-C5 subgroup to 51%+/-19 for T7-L3 subgroup. The average percent predicted of all participants for MIP was 74%+/-30 and for MEP was 37%+/-21. In patients with complete motor lesion, the correlation with the level of injury was stronger for MEP (r=0.81, P<0.0001; r (2)=0.65) than for MIP (r=0.62, P=0.004; r (2)=0.38). No correlation was found among incomplete motor lesion patients. CONCLUSIONS: The linear regression equations for the relationship of percent predicted MIP or MEP to level of injury are applicable only to complete motor lesions and may be useful to establish normative association between them.

Sampling for collection of central line-day denominators in surveillance of healthcare-associated bloodstream infections.

OBJECTIVE: To determine the feasibility of estimating the number of central line-days at a hospital from a sample of months or individual days in a year, for surveillance of healthcare-associated bloodstream infections. DESIGN: We used data reported to the National Nosocomial Infections Surveillance system in the adult and pediatric intensive care unit component for 1995-2003 and data from a sample of hospitals' daily counts of device use for 12 consecutive months. We calculated the percentile error as the central line-associated bloodstream infection percentile based on rates per line-days minus the percentile based on rates per estimated line-days. SETTING AND PARTICIPANTS: A total of 247 hospitals were used for sampling whole months and 12 hospitals were used for sampling individual days. RESULTS: For a 1-month sample of central line-days data, the median percentile error was 3.3 (75th percentile, 7.9; 90th percentile, 15.4). The percentile error decreased with an increase in the number of months sampled. For a 3-month sample, the median percentile error was 1.4 (75th percentile, 4.3; 95th percentile, 8.3). Sampling individual days throughout the year yielded lower percentile errors than sampling an equivalent fraction of whole months. With 1 weekday sampled per week, the median percentile error ranged from 0.65 to 1.40, and the 90th percentile ranged from 2.8 to 5.0. Thus, for 90% of units, collecting data on line-days once a week provides an estimate within +/-5 percentile points of the true line-day rate. CONCLUSION: Sample-based estimates of central line-days can yield results that are acceptable for surveillance of healthcare-associated bloodstream infections.

Cholinergic bronchomotor tone and airway caliber in tetraplegic patients.

STUDY DESIGN: Case-control study. OBJECTIVES: To evaluate the resting airway caliber in subjects with tetraplegia; to define the participation of cholinergic innervation in this condition; and to determine if baclofen modifies this pattern. SETTING: A rehabilitation hospital, Brasília, Brazil. METHODS: We studied 18 tetraplegic patients, with complete motor loss between C4 and C8, and 18 healthy control subjects by measuring airway conductance, before and after inhaled ipratropium bromide. RESULTS: At baseline, the pulmonary function parameters revealed mild-to-moderate restrictive impairment in tetraplegic patients as defined by decreases in total lung capacity and predicted percent of slow vital capacity. The average baseline specific airway conductance (sGaw) was less in tetraplegic patients (0.25+/-0.11) than in the control group (0.41+/-0.10 l/s/cm H(2)O) (P<0.0001). All patients had improved post-bronchodilator sGaw >or=40% compared with only four of the 18 controls (P<0.001). The average increase for tetraplegic patients was 235% (+/-93) versus 25% (+/-24) for controls (P<0.0001). Analysis of variance for repeated measurements showed significant difference in sGaw between the control and spinal cord injury (SCI) groups (P<0.0001) following bronchodilator challenge, but found no difference for total gas volume. No difference for mean basal sGaw and bronchodilator challenge was encountered comparing tetraplegic patients using baclofen to those not using it. CONCLUSIONS: Cervical SCI patients have a reduced baseline conductance compared to controls. Marked improvement occurs after an inhaled anticholinergic drug. This behavior was not affected by the use of baclofen. The study adds support to the hypothesis of an increased cholinergic bronchomotor tone in tetraplegic patients.

Predictive factors of functional gain in long-term stroke survivors admitted to a rehabilitation programme.

PRIMARY OBJECTIVE: To assess factors that may influence functional gain of patients with chronic sequelae of stroke. RESEARCH DESIGN: Prospective study of 290 stroke patients consecutively admitted to a rehabilitation setting. METHODS AND PROCEDURES: Functional Independence Measure Scale (FIM) was used to assess functional capacity. Functional improvement registered during hospitalization (FIM-gain score) was compared to demographic data, stroke sub-type, vascular risk factors, motor deficit, visual hemineglect, aphasia, level of response and sphincter control. FIM-gain score was classified as high-gain (=22) and low-gain (<22). MAIN OUTCOMES AND RESULTS: Two hundred and fifty-two patients who had no prior rehabilitation and were capable of completing the rehabilitation programme were studied (average age 58.4+/-13.9 years; 42.9% women). The mean time from stroke onset to admission was 271.5 days. Average FIM score at admission was 58.8 and at discharge was 81.6. Average FIM Gain was 23.6. The 38% patients admitted later than 6 months after stroke had an average FIM Gain of 19 vs 26 for patients admitted prior to 6 months. Significant predictors of functional improvement were time from stroke onset, age, sitting balance and level of responsiveness. CONCLUSION: The functional improvement scores in persons with stroke beginning a rehabilitation programme at a later stage are 73% of the scores obtained by patients beginning treatment in the first 6 months. FIM score improvement can be predicted by time since stroke onset, age, sitting balance and level of responsiveness.

Involvement of 5-HT receptors in the regulation of food intake in Siberian hamsters.

The Siberian hamster provides a physiological model for understanding the hypothalamic control of energy metabolism as it undergoes annual photoperiod-regulated cycles of body weight (i.e. fattening in summer, and catabolism of fat stores in winter). As a first step to investigate whether enhanced serotonergic (5-HT) tone might underlie the catabolic processes in short days, we investigated whether serotonergic stimulation can produce catabolic actions in fat hamsters housed in long days. Acute treatment with the serotonin reuptake inhibitor (+/-) fenfluramine (8 mg/kg, i.p.) produced a prolonged, dose-dependent reduction in food intake in both photoperiods. Behavioural observations and radiotelemetry analyses revealed that this anorectic effect of fenfluramine was associated with short-term increases in locomotor activity and in core body temperature. In a subsequent series of studies, hamsters were pretreated with the 5-HT2C receptor antagonist SB242084 (4 mg/kg, i.p.). This 5-HT2C receptor antagonist completely blocked the anorectic actions of fenfluramine, but did not decrease the hyperthermia or hyperlocomotion induced by fenfluramine; thus, the anorectic actions of fenfluramine probably reflect actions via the 5-HT2C receptor. Consistent with these observations, treatment of hamsters with the 5-HT2C receptor agonist VER 3323 (10 mg/kg, i.p.) or the 5-HT1B/2C receptor agonist mCPP (3 mg/kg, i.p.) reduced food intake. The response to manipulation of serotonergic pathways was not affected by the ambient photoperiod in any of these studies. We conclude that the anorectic actions of fenfluramine are not an indirect consequence of serotonergic actions on arousal pathways, and that its actions on feeding in the Siberian hamster are most likely to be mediated by the 5-HT2C receptor.

The expression patterns of mRNA-encoding stem cell factor, internal stem cell factor and c-kit in the prepubertal and adult porcine ovary.

The receptor, c-kit, and its ligand, stem cell factor (SCF), are important regulators of ovarian follicle growth and development. The aim of this study was to identify the sites of expression of mRNA for c-kit and SCF in prepubertal and mature (pregnant and non-pregnant) animals. Ovaries were recovered from prepubertal animals, non-pregnant sows and five sows at approximately 3 months of gestation. Ovine SCF and c-kit DNA were cloned into plasmid vectors to produce RNA probes. Expression of mRNA encoding SCF and c-kit were detected via in situ hybridization. Both mRNA were detected throughout ovaries from all animals. This study provides evidence that the growth-factor complex is required throughout follicle development, and also for continued maintenance of the corpus luteum (CL) in the mature animal. SCF mRNA was localized to the granulosa cell layer and was also extensively expressed in endothelial tissue and throughout the CL. c-kit mRNA was detected in the theca layer, oocytes and also in CL. In conclusion, expression of SCF and c-kit mRNA in granulosa and theca cells, respectively, indicate an important interaction between somatic cells throughout follicle development and that in the mature animal, SCF and c-kit potentially have a role in maintaining progesterone secretion by the CL. The observations of continued expression of SCF and c-kit throughout development suggest that there may be differences in the role of this receptor-ligand complex between large mono- vs. poly ovulatory species, such as the pig.

Schistosoma mansoni myelopathy: clinical and pathologic findings.

Thirteen patients with Schistosoma mansoni myelopathy are reported. Neurologic syndromes included acute areflexic flaccid paraplegia (three), thoracic myelopathy with hyperreflexia and Babinski sign (six), and a cauda equina syndrome (four). Inflammatory granulomas and a schistosome worm in a leptomeningeal vein of the spinal cord were observed in the one patient coming to necropsy.

Secretin: hypothalamic distribution and hypothesized neuroregulatory role in autism.

1. This study aims (1) to determine whether secretin is synthesized centrally, specifically by the HPA axis and (2) to discuss, on the basis of the findings in this and previous studies, secretin's possible neuroregulatory role in autism. 2. An immunocytochemical technique with single-cell resolution was performed in 12 age/weight-matched male rats pretreated with stereotaxic microinjection of colchicine (0.6 microg/kg) or vehicle into the lateral ventricle. Following 2-day survival, rats were anesthetized and perfused for immunocytochemistry. Brain segments were blocked and alternate frozen 30-microm sections incubated in rabbit antibodies against secretin, vasoactive intestinal peptide, glucagon, or pituitary-adenylate-cyclase-activating peptide. Adjacent sections were processed for Nissl stain. Preadsorption studies were performed with members of the secretin peptide family to demonstrate primary antibody specificity. 3. Specificity of secretin immunoreactivity (ir) was verified by clear-cut preadsorption control data and relatively high concentrations and distinct topographic localization of secretin ir to paraventricular/supraoptic and intercalated hypothalamic nuclei. Secretin levels were upregulated by colchicine, an exemplar of homeostatic stressors, as compared with low constitutive expression in untreated rats. 4. This study provides the first direct immunocytochemical demonstration of secretinergic immunoreactivity in the forebrain and offers evidence that the hypothalamus, like the gut, is capable of synthesizing secretin. Secretin's dual expression by gut and brain secretin cells, as well as its overlapping central distribution with other stress-adaptation neurohormones, especially oxytocin, indicates that it is stress-sensitive. A neuroregulatory relationship between the peripheral and central stress response systems is suggested, as is a dual role for secretin in conditioning both of those stress-adaptation systems. Colchicine-induced upregulation of secretin indicates that secretin may be synthesized on demand in response to stress, a possible mechanism of action that may underlie secretin's role in autism.

Secretin activates visceral brain regions in the rat including areas abnormal in autism.

1. The aim of this study was to determine whether central networks are involved in the presumptive behavioral and autonomic regulatory actions of secretin, a gut hormone that has been reported to have ameliorative effects in autistic children. 2. Central neural responses monitored by regional c-fos gene expression were examined in response to intracerebroventricular secretin injection in awake, freely-moving Sprague-Dawley rats. Tissue sections were incubated in an antibody to the c-fos gene product, Fos, and processed immunohistochemically. 3. Qualitative differences in Fos immunoreactivity in stress adaptation and visceral representation areas of the brain were observed between secretin- and vehicle-infused age-matched pairs (n = 4 pairs). Secretin-activated regions include the area postrema, dorsal motor nucleus, medial region of the nucleus of the solitary tract and its relay station in the lateral tegmentum, locus ceruleus, ventral periaqueductal gray, periventricular thalamic nucleus, paraventricular hypothalamus magnocellularis, medial and central amygdala, lateral septal complex as well as ependymal and subependymal nuclei lining the third ventricle. Specific areas of the cerebral cortex were heavily labeled in secretin-treated rats, as compared to controls: the medial bank of the anterior prefrontal cortex, orbitofrontal cortex, the piriform cortex. and the anterior olfactory nucleus. Secretin attenuated Fos immunoreactivity in the dorsal periaqueductal gray, intralaminar thalamus, medial parvicellular compartment of the hypothalamus, supraoptic nucleus of the hypothalamus, lateral amygdala, motor cortex, and the somatosensory and association areas of the parietal cortex. 4. Secretin alters the activity of structures involved in behavioral conditioning of stress adaptation and visceral reflex reactions. This study predicts a possible cellular mechanism, activation of third ventricular ependymal and subependymal cells, as well as central regulatory actions of secretin. The physiological effects of secretin on behavioral, endocrine, autonomic and sensory neuronal activation patterns, together, contribute to central c-fos activation. Secretin alters the activity of structures involved in behavioral conditioning of stress adaptation and visceral reflex reactions. This study predicts a possible cellular mechanism, activation of third ventricular ependymal and subependymal cells, and central regulatory actions of secretin. The physiological effects of secretin on behavioral, endocrine, autonomic and sensory neuronal activation patterns, together, contribute to central c-fos activation. These findings mandate further investigation of secretin as a brain/gut stress regulatory hormone.

American trypanosomiasis (Chagas' disease): an unrecognised cause of stroke.

BACKGROUND: American trypanosomiasis, known as Chagas' disease (CD) is a major cause of cardiomyopathy in South America. Irreversible damage to the heart can appear 10 to 20 years after chagasic infection. The frequency of cerebrovascular complications in chronic CD is unknown. OBJECTIVES: To describe a group of patients with chronic or latent CD affected by ischaemic stroke and identify predictive variables for stroke in CD patients. PATIENTS AND METHODS: Retrospective case series of stroke patients with CD was studied using a cross sectional, descriptive design. CD was confirmed by positive immunofluorescence and haemaglutination serology. Data were collected on age, sex, vascular risk factors, previous history of CD, diagnostic stroke subtype, electrocardiograph and echocardiography findings. Frequency of vascular risk factors were compared with a control group of 239 non-chagasic stroke patients. RESULTS: 136 consecutive CD stroke patients, mean age 56 years, 72 women and 64 men were identified. Vascular risk factors were observed in 81.6% of CD patients. Hypertension (70.29% versus 51.47%; p=0.0004), diabetes mellitus (15.9% versus 6.61%; p=0.0143), and tobacco use (53.98% versus 30.88%; p=0.00002) were significantly less frequent in the CD stroke group. Cardiomyopathy was significantly higher in CD stroke patients (45.58% versus 24.69%; p=0.00005). Abnormal electrocardiograms was observed in 82% of chagasic patients (right bundle branch block 39.5%, left anterior fascicular block 35.8%). Left ventricle (LV) diastolic dysfunction (61.47%), LV systolic dysfunction (51.18%), congestive cardiomyopathy (29.92%), and apical aneurysm (15.74%) were the most frequent echocardiographic findings. Aetiologies were cardioembolism (52.2%), undetermined (36.76%), atherothrombotic (8.82%), and small vessel stroke (2.2%). A diagnosis of CD was established after presentation with stroke in 38.23% of the patients. CONCLUSIONS: CD should be included in the differential diagnosis of stroke in patients of South American origin.

The membrane-anchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis.

Matrix metalloproteinases (MMPs) are essential for proper extracellular matrix remodeling. We previously found that a membrane-anchored glycoprotein, RECK, negatively regulates MMP-9 and inhibits tumor invasion and metastasis. Here we show that RECK regulates two other MMPs, MMP-2 and MT1-MMP, known to be involved in cancer progression, that mice lacking a functional RECK gene die around E10.5 with defects in collagen fibrils, the basal lamina, and vascular development, and that this phenotype is partially suppressed by MMP-2 null mutation. Also, vascular sprouting is dramatically suppressed in tumors derived from RECK-expressing fibrosarcoma cells grown in nude mice. These results support a role for RECK in the regulation of MMP-2 in vivo and implicate RECK downregulation in tumor angiogenesis.

Appositions between cocaine and amphetamine-related transcript- and gonadotropin releasing hormone-immunoreactive neurons in the hypothalamus of the Siberian hamster.

Recent in vitro studies have provided evidence that cocaine and amphetamine-related transcript (CART) pathways in the hypothalamus mediate the effects of leptin upon gonadotropin releasing hormone (GnRH) secretion. The aim of the current study was to use dual label immunofluorescence to investigate the anatomical basis of such a pathway. CART-ir processes were found extensively in regions where GnRH cell bodies where located. Analysis using confocal microscopy showed that the majority of GnRH neurons (62%) had close appositions from CART-ir processes. The proportion of GnRH-ir perikarya with CART-ir appositions was significantly higher (P<0.05) in neurons located in the diagonal band of Broca (70%) compared to those more caudally located in the preoptic area (53%). This anatomical evidence for close appositions between CART-ir processes and GnRH cell bodies supports the hypothesis that one mechanism by which leptin causes its effect on the GnRH pulse generator is indirectly via CART neurons, thus allowing information about nutritional status and body fat stores to be conveyed to the reproductive system.

Identification of a heregulin binding site in HER3 extracellular domain.

HER3 (also known as c-Erb-b3) is a type I receptor tyrosine kinase similar in sequence to the epidermal growth factor (EGF) receptor. The extracellular segment of this transmembrane receptor contains four domains. Domains I and II are similar in sequence to domains III and IV, respectively, and domains II and IV are cysteine-rich. We show that the EGF-like domain of heregulin (hrg) binds to domains I and II of HER3, in contrast to the EGF receptor, for which prior studies have shown that a construct consisting of domains III and portions of domain IV binds EGF. Next, we identified a putative hrg binding site by limited proteolysis of the recombinant extracellular domains of HER3 (HER3-ECD(I-IV)) in both the presence and absence of hrg. In the absence of hrg, HER3-ECD(I-IV) is cleaved after position Tyr(50), near the beginning of domain I. Binding of hrg to HER3-ECD(I-IV) fully protects position Tyr(50) from proteolysis. To confirm that domain I contains a hrg binding site, we expressed domains I and II (HER3-ECD(I-II)) and find that it binds hrg with 68 nm affinity. These data suggest that domains I and II of HER3-ECD(I-IV) act as a functional unit in folding and binding of hrg. Thus, our biochemical findings reinforce the structural hypothesis of others that HER3-ECD(I-IV) is similar to the insulin-like growth factor-1 receptor (IGF-1R), as follows: 1) The protected cleavage site in HER3-ECD(I-IV) corresponds to a binding footprint in domain I of IGF-1R; 2) HER3-ECD(I-II) binds hrg with a 68 nm dissociation constant, supporting the hypothesis that domain I is involved in ligand binding; and 3) the large accessible surface area (1749 A) of domain L1 of IGF-1R that is buried by domain S1, as well as the presence of conserved contacts in this interface of type 1 RTKs, suggests that domains L1 and S1 of IGF-1R function as a unit as observed for HER3-ECD(I-II). Our results are consistent with the proposal that HER3 has a structure similar to IGF-1R and binds ligand at a site in corresponding domains.