Genetic signatures of high- and low-risk aberrant crypt foci in a mouse model of sporadic colon cancer

To determine whether cancer risk is related to histopathological features of preneoplastic aberrant crypt foci (ACF), gene expression analysis was performed on ACF from two mouse strains with differing tumor sensitivity to the colonotropic carcinogen, azoxymethane. ACF from sensitive A/J mice were considered at high risk, whereas ACF from resistant AKR/J mice were considered at low risk for tumorigenesis. A/J and AKR/J mice received weekly injections of azoxymethane (10 mg/kg body weight), and frozen colon sections were prepared 6 weeks later. Immunohistochemistry was performed using biomarkers associated with colon cancer, including adenomatous polyposis coli, beta-catenin, p53, c-myc, cyclin D1, and proliferating cell nuclear antigen. Hyperplastic ACF, dysplastic ACF, microadenomas, adjacent normal-appearing epithelium, and vehicle-treated colons were laser captured, and RNA was linearly amplified (LCM-LA) and subjected to cDNA microarray-based expression analysis. Patterns of gene expression were identified using adaptive centroid algorithm. ACF from low- and high-risk colons were not discriminated by immunohistochemistry, with the exception of membrane staining of beta-catenin. To develop genetic signatures that predict cancer risk, LCM-LA RNA from ACF was hybridized to cDNA arrays. Of 4896 interrogated genes, 220 clustered into six broad clusters. A total of 226 and 202 genes was consistently altered in lesions from A/J and AKR/J mice, respectively. Although many alterations were common to both strains, expression profiles stratified high- and low- risk lesions. These data demonstrate that ACF with distinct tumorigenic potential have distinguishing molecular features. In addition to providing insight into colon cancer promotion, our data identify potential biomarkers for determining colon cancer risk in humans.     

Antibiotic use for upper respiratory tract infections: how well do pediatric residents do?

BACKGROUND: Antibiotics are often used inappropriately for the treatment of upper respiratory tract infections in children, and the emergence of resistant bacteria is a growing public health concern. OBJECTIVE: To assess awareness and compliance with the Centers for Disease Control and Prevention (Atlanta, Ga) and American Academy of Pediatrics (Elk Grove Village, Ill) principles for judicious antibiotic use for upper respiratory tract infections among residents from a sample of pediatric residency programs in the mid-Atlantic region of the United States. PARTICIPANTS AND METHODS: Residents at the participating programs were requested to complete a survey questionnaire. RESULTS: Of the 524 pediatric residents surveyed, 74% (388 participants) completed the questionnaire. Familiarity with the principles increased with a year of training; 16%, 36%, and 50% of first-year (PL1), second-year (PL2), and third- or fourth-year (PL3/PL4) residents, respectively, had heard or read about the principles (chi(2)(trend); P<.001). In response to a direct question about the use of antibiotics for an otherwise well, afebrile 18-month-old child with purulent rhinorrhea, 29%, 25%, and 15% of PL1, PL2, and PL3/PL4 residents, respectively, would prescribe antibiotics within 10 days of onset of illness (chi(2)(trend); P =.008). A significant difference was found between PL1 vs PL3/PL4 participants (difference = 20%; 95% CI = 3%-26%). If the same infant had a temperature of 38.8 degrees C, then 63%, 45%, and 47% of PL1, PL2, and PL3/PL4 residents, respectively, would prescribe antibiotics (chi(2)(trend); P =.008). CONCLUSIONS: Awareness among pediatric residents about the judicious use of antibiotics for upper respiratory tract infections is often lacking, and inappropriate use of antibiotics for this condition continues to be prevalent. This was especially noted among PL1 residents, with an improving trend noted with increasing years of training.     

Molecular and biochemical evidence for the presence of type III adenylyl cyclase in human platelets

The isoform(s) of adenylyl cyclase (AC) present in human platelets has not been identified, and evidence supporting a role for AC in platelet aggregation is equivocal. We recently characterized deaggregation as an active component of the platelet aggregation response that may be an important determinant of the extent and duration of aggregation. G(i)-coupled receptors are linked to the inhibition of AC and are targets of antiplatelet drugs. They also affect platelet aggregation by modulating deaggregation, suggesting a role for AC in modulating this response. The purpose of this study was to identify the AC isoform(s) present in human platelets and to identify its physiological modulators. RT-PCR screening of platelet, buffy coat layer cell and bone marrow megakaryocyte cDNA, and Western blot analysis with AC type III (AC-III) antibodies identified AC-III in platelets and in megakaryocytes. Human platelet AC-III was cloned and expressed in HEK293 cells and its characteristics compared to native platelet AC. Both platelet AC and cloned AC-III required Mg(2+) for activity, were insensitive to Ca(2+) and were G(s)- and G(i)-coupled. Zn(2+) and SQ22536 inhibited platelet AC activity. The affinity of SQ22536 was increased with Mg(2+)-related stimulation of AC, while that of Zn(2+) was unchanged, which is consistent with a non-competitive interaction between the two metal ions on AC. The Zn(2+) chelator TPEN reversed the inhibitory effects of Zn(2+). This study identified AC-III as the predominant AC isoform in human platelets, the activity of which may affect the extent and duration of the net aggregation response by modulating deaggregation.     

Agonist concentration-dependent differential responsivity of a human platelet purinergic receptor: pharmacological and kinetic studies of aggregation, deaggregation and shape change responses mediated by the purinergic P2Y1 receptor in vitro

Platelet shape change (SC), aggregation and deaggregation responses are integral components of hemostasis that are elicited and modulated in vivo by the simultaneous activation of several membrane receptors. Selective activation of the purinergic P2Y1 receptor in vivo elicits a sustained SC and a small, transient aggregation response that is reversed rapidly by a robust deaggregation response (Platelets 2003; 14: 89). Using a kinetics-based turbidimetric approach to study the modulation of these concurrent components of human platelet responses, we demonstrate that these P2Y1 receptor-related responses and a number of their kinetic and steady-state characteristics are differentially elicited and modulated. P2Y1 receptor agonist concentrations that elicited aggregation (pEC50 for ADP, 2-MeSADP; 5.88, 6.69) were 10-fold greater than those that elicited SC (7.33, 7.67). The magnitude of the aggregation response was agonist concentration-dependent, saturable and was associated with an agonist concentration-dependent deceleration of the deaggregation response. Gi-coupled receptor (alpha 2A-adrenoceptor, EP3 and P2Y12 receptors) agonists also enhanced aggregation through deceleration of the deaggregation response, and an inhibitor of PI3K activity (wortmannin) inhibited aggregation through acceleration of the deaggregation response. Neither treatment affected the extent or the kinetics of the SC response. The aggregation but not the SC response was rapidly desensitized by P2Y1 receptor activation by ADP. The affinity of the presence of a single P2Y1 receptor subtype. The differential characteristics and modulation of the SC and aggregation responses by a single receptor support the idea that different signaling pathways activated at different occupancy states of the same receptor underlie the two responses. P2Y1 receptor-mediated platelet aggregation and SC responses provide a convenient model for studying the phenomenon of agonist-directed signaling by differential occupancy of the same membrane receptor.     

Change in epidemiology of health care-associated infections in a neonatal intensive care unit

BACKGROUND: Gram-negative rods (GNR) and species are important causes of health care-associated infections in neonatal intensive care units (NICUs). In prior reports, Gram-positive cocci (GPC) have been identified as the most common pathogens causing nosocomial infections in NICUs. OBJECTIVE: To describe the epidemiology of health care-associated infections in a Level III NICU at a free-standing children's hospital. METHODS: All health care-associated infections in neonates from August 1996 to July 2001 were analyzed. Data were collected prospectively by standard surveillance protocols and nosocomial infection site definitions from the National Nosocomial Infection Surveillance system of the Centers for Disease Control and Prevention. RESULTS: During the 5-year study period, 665 pathogens caused 640 infections in the NICU. GNR were the most common pathogens isolated. Of the 665 pathogens 284 (43%) were GNR, followed by 223 (33.5%) GPC, 106 (16%) fungi and 52 (8%) others. spp. were the most common GNR isolated. Ceftazidime resistance was present in 56 of 81 (69%) isolates. Bloodstream and lower respiratory tract infections were the most common sites of infection. More than one-half of the pathogens (388 of 665) were isolated from neonates weighing < or =1000 g. CONCLUSIONS: There is a change in the epidemiology of health care-associated infections in our NICU with a predominance of GNR. This change can impact choice of antimicrobials for the empiric treatment of health care-associated infections in high risk neonates.     

Surgery in Singapore

Since Singapore became an independent nation about 50 years ago, remarkable progress has been made in the development and organization of the health care system, specialty services, graduate training, and medical research. The primitive health care service under the British colonial service before the Second World War which was further ravaged in the 1940s by the Japanese occupation has been radically transformed in a short period. Singapore has a modern health care service, excellent hospital facilities, an affordable health financing system, adequate medical manpower including specialists, and a postgraduate organization for the training of medical specialists. The advancement in surgical services has reached a high level where all forms of modern surgery, including laparoscopic and endoscopic operations, are routine and major bone and joint replacements, heart and liver transplantations, and complex neurosurgical and craniofacial operations are frequently carried out.     

Aberrant transforming growth factor-beta signaling in azoxymethane-induced mouse colon tumors

Alterations in the transforming growth factor-beta (TGF-beta) pathway are implicated in the pathogenesis of colorectal cancer. We hypothesize that alterations in the TGF-beta pathway contribute to differential sensitivity of mice to the colon carcinogen azoxymethane (AOM). A/J (sensitive) and AKR/J (resistant) mice were injected intraperitoneally with AOM (10 mg/kg of body weight once a week for 6 wk). Twenty-four weeks after AOM exposure, mutational analysis of TGF-beta type II receptor (TbetaR-II) from normal colons and from tumors showed no AOM-induced alterations. A significant decrease (1.5-fold, P < 0.05) in TbetaR-II mRNA levels, however, was found in A/J tumors with the RNase protection assay. Immunofluorescence of TbetaR-II showed marked loss of staining in A/J tumors. The RNase protection assay and sequence analysis of the downstream signaling molecule Smad3 revealed no carcinogen-induced alterations in either strain. To gain further insight into the functionality of the pathway, expression of TGF-beta, TGF-beta type I receptor (TbetaR-I), and several downstream targets of TGF-beta signaling, including Smad7, c-myc, and p15, was examined. Although no alterations in TGF-beta, TbetaR-I, or Smad7 were found in tumors, a significant increase in c-myc expression (2.5-fold, P < 0.05 ) and a significant decrease in p15 expression (4.5-fold, P < 0.05 ) were noted. Concomitant repression of TbetaR-II and overexpression of c-myc may render epithelial cells insensitive to TGF-beta-mediated growth arrest, a possibility that also is suggested by this model. The significant decrease in p15 expression in tumors provides additional evidence that TGF-beta signaling may be markedly attenuated during colon tumorigenesis.