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If you're searching for an exciting place to call home, look no further than colorful New Jersey! Its cities and suburbs, townships and boroughs are known to be among the fastest-growing in the United States.
See what each of these New Jersey towns and cities can offer:
- Secaucus – The Jewel of New Jersey's Meadowlands
Secaucus is a suburban community located just five miles from Times Square in Manhattan. Its population grew by 24.3% from 16,260 in 2010 to 20,215 in July 2017. Several major corporate headquarters and a distribution center for Northern New Jersey and Manhattan fuel its economy. The presence of the distribution center resulted in another booming business in Secaucus – warehouse outlets.
- Hoboken – The Mile Square City
Hoboken is a city with a rich heritage, an active music scene, and special amenities that make it an ideal place to move to. US Census Bureau stats show a 10.2% increase in its population, from 50,006 in 2010 to 55,131 in 2017.
Hoboken is famous for being the birthplace of legendary crooner, Frank Sinatra. It is also home to the Hoboken Terminal, a major transportation hub serving New York City and the rest of the region. Thanks to its attractions and conveniences, Hoboken took the top spot in Niche.com's 2018 Best Places for Millennials in New Jersey.
- Edgewater – A riverside borough
The promise of peaceful waterfront living in Edgewater is a big draw for those who desire to live near the water but not far from city conveniences. Thriving communities dot the Hudson River banks, with many homes boasting spectacular views.
Edgewater has seen a 7.4% uptick in its population in less than a decade from 11,516 in 2010 to 12,368 in 2017. This growth can be attributed in part to its proximity to New York, which is just around 11 miles away.
- Raritan – A borough with highly rated schools, an active nightlife
Raritan in Somerset County is great for raising a family. Your children will be attending highly rated schools under the Raritan Regional School District.
Residents take on a healthy lifestyle and enjoy what Raritan can offer, from outdoor activities to an active nightlife. This is why many young professionals prefer living in Raritan. They are what constitute a 2017 population of 8,011 – a 16.4% rise from 2010's 6,882.
- Harrison – A town with a youthful vibe
The town of Harrison in Hudson County occupies just 1.31 square miles, with a steadily growing population consisting mostly of young people. From 13,551 in 2010, Harrison's population has grown by over 30% to 17,643 in 2017. There are many apartment complexes in the area where students from nearby universities live. The town is also only 16.5 miles from The Big Apple – a big advantage for residents who work in the city.
Harrison is the fifth best place to live in Hudson County, according to Niche.com because of its youthful vibe and urban feel.
- West New York – A town with future-ready schools
The origin of West New York's name is quite self-explanatory, being a suburb in the New Jersey Palisades located west of New York City. Around 80% of residences here are mostly apartments while the remaining 20% are single-family homes. The population here grew up by 9.1% to 54,227 in 2017 from 49,708 in 2010.
West New York's school district is considered high-performing by the state of New Jersey. Schools under this district have been declared future-ready by the state's Department of Education.
- Montvale – A borough with thriving economic opportunities
Montvale is one of 26 boroughs established in Bergen County in 1894. Since that time, it has grown in population size and economic opportunities. In 2017, Montvale's population was 8,724 – an 11.2% increase from 2010's 7,844. The thriving industries here are in healthcare and social assistance (15.4%); professional, scientific, and technological services (13.4%); and manufacturing (12.4%).
- Jersey City – Culturally diverse boom town
Jersey City has some of the most spectacular waterfront views, especially in its eastern portion which faces the Hudson River. From Liberty State Park, one has a clear view of Manhattan's skyline and the Statue of Liberty.
The city saw a 9.3% rise in population , from 247,597 in 2010 to 270,753 in 2017. It has an efficient transportation system, making it easy to get to New York City, which is four miles away. Jersey City hosts booming industries in finance and tech services and is known to be a culturally diverse.
- Palisades Park – Home to Koreatown and well-known universities
Palisades Park is another New Jersey borough with an economy fueled primarily on industries related to retail, tech services, and healthcare. From 19,622 in 2010, the population of Palisades Park has grown by 7% to 20,988 in 2017.
Palisades Park is home to Koreatown, a center for Korean culture in New Jersey. It is also close to well-known universities like New York University and Columbia University.
- Rahway – Diverse and affordable
Rahway City in Union County has seen a population growth of 10.2% between 2010 and 2017 from 27,346 to 30,131. It's home to diverse peoples and has more affordable housing options. The Union County Performing Arts Center, the venue for the annual Rahway International Film Festival, is found here. Rahway is just 24 miles away from New York City.
Do you have plans of moving to New Jersey? You can get in touch with me, Janine Rose, to explore your real estate options. Call me today at 908.229.6252 or send an email to Janine(at)janinerose(dotted)com.


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subset: commoncrawl


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Research Output per year
Personal profile
Professor Jac Birt joined the UWA Business School in January 2018. Previously she was employed at the UQ Business School, Monash Business School, the ANU and the University of Melbourne. She is a Professor of Accounting and also the Head of Department - Accounting and Finance for the UWA Business School.
Jac is a member of several external boards including AFAANZ, the IAESB Consultative Advisory Group and BEWA (Business Educators Western Australia). She is a Deputy Editor at the Accounting & Finance Journal and an Associate Editor at the Accounting Research Journal.
Jac's research is predominantly in international accounting and accounting education. In international accounting she has published in areas such as segment reporting, voluntary disclosures and value relevance. In accounting education she has published in areas such as XBRL, ICT use in accounting, international student learning and blended learning. She has published over 20 refereed journal articles including Abacus, Journal of Business Ethics, Sport Management Review, Australian Journal of Management, Accounting and Finance, Accounting Research Journal, Australian Journal of Adult Learning, Australian Accounting Review, Accounting in Europe, International Journal of Pedagogy and Curriculum and Accounting Education. She is currently working on projects including accreditation issues, team-based learning, internet reporting, pension plans, accounting regulation and insurance contracts.
Jac is also the main author on the leading Wiley Australian accounting textbook - "Accounting: Business Reporting for Decision Making" 6th edition.
Jacqueline is currently supervising PhD students in the following areas:
* Value relevance of management earnings forecasts
* The changing landscape of management earnings forecasts: The determinants and implications of the increase in range forecasts
* Economic growth implications for environmental quality indicators: A review and empirical analyses for dynamic endogenous Environmental Kuznets Curve
* Demystifying the process of accounting for groups:Utilising Adaptive Learning Technologies
Education/Academic qualification
Commerce, PhD, Australian National University
Education, Bachelor of Education, Melbourne College of Advanced Education
Business, Master of Business, Royal Melbourne Institute of Technology University
Accounting, Bachelor of Business , Royal Melbourne Institute of Technology University
External positions
Committee member, Business Educators Western Australia (BEWA)1 Apr 2018 → …
Board member of AFAANZ
Board member of IAESB
- Financial accounting
- Accounting Education
Fingerprint Dive into the research topics where Jacqueline Birt is active. These topic labels come from the works of this person. Together they form a unique fingerprint.
Recent external collaboration on country level. Dive into details by clicking on the dots.
Research Output 2018 2019
- 2 Article
Do Risk Disclosures Relating to the Use of Financial Instruments Matter? Evidence from the Australian Metals and Mining SectorThai, K. H. P. & Birt, J., 12 Dec 2019, In : International Journal of Accounting. 1950017.
Research output: Contribution to journal › Article
Standard Business Reporting (SBR) Adoption in Australia, Critically Acclaimed, Box Office Flop: Constructivist and Ecological Rationalities in Information Systems (IS) AdoptionPerdana, A., Robb, A., Rohde, F. & Birt, J., 2018, In : Australasian Journal of Information Systems. 22, 29 p.
Research output: Contribution to journal › Article
Projects 2018 2020
Projects per year
Inspiring Future-Oriented Graduates with Innovative Curriculum Design: Accounting in the Digital Age
31/01/19 → 30/06/20
Project: Research
The effectiveness of teamwork assessment tasks
Bayne, L., Birt, J. & Schonfeldt, N.
Accounting & Finance Association of Australia & New Zealand (AFAANZ)
1/07/18 → 30/06/19
Project: Research


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It is better for you to use electric heating instead of gas heating for so many reasons that it is now becoming standard practice for more homes. Here are some reasons why electric heating is now the preferred option:
Electric heating is a practical solution that requires much less maintenance than an oil or gas heater. It has the particular advantage of requiring very little installation work compared to other heating modes. It is therefore particularly recommended when the installation of a central heating in the housing is too complicated. It is a non-messy option that makes it possible to not lose space unnecessarily since with the electric, there is no fuel to store!
Before, electric heating suffered rather from a bad reputation. In particular, it could be blamed for diffusing a uneven heat, and for presenting too frequent variations of temperature. Today, the technological advances made by the major manufacturers of electric heating offer optimal heat without any loss. In terms of efficiency, electric heaters are now really powerful.
Electric heating also has the advantage of being healthier than some of its competitors, since it requires no combustion, and therefore generates no smoke in the housing.
On the energy side, the price of electricity is lower than gas, and there are few maintenance costs. Most electric heaters now offer programming (comfort and eco modes) but also many intelligent functions that will allow you to enjoy the best comfort while achieving energy savings. These features can save up to 45% on your heating bill.
The intelligent control of your electric heating offers you an incomparable quality of comfort while allowing you to realize considerable savings. Totally invisible, it operates in all discretion. Integrated with your new radiator, it uses a presence detector to optimize your energy consumption according to your lifestyle. After having memorized your habits automatically, it schedules the temperature according to your displacements. For example, when you are at work, it reduces the temperature itself! Then, he anticipates your return to guarantee you the ideal temperature as soon as you come home.
This heater is able to adapt and react as quickly as possible in the event of a change in the environment. Thus, the presence detector acts and modifies the temperature setpoint.
By replacing your old convectors with smart-driven electric heaters, you can save money and increase comfort. Remember also that for the installation of a heating regulation system, you can benefit from a tax credit. Switching over to electric heating is easy. Depending on your home, this specialist will establish a quote tailored to your needs.
On the energy side, the price of electricity is lower than gas, and there are few maintenance costs. Most electric heaters now offer programming (comfort and eco modes) but also many intelligent functions that will allow you to enjoy the best comfort while achieving energy savings. These features can save up to 45% on your heating bill.
Note that if any fire hazard from your electric heater ensues, it is considered foolhardy to jump on it by oneself. Contacting an electrician in Altamonte Springs, Fl is the best choice. There are extremely skilled electricians in Altamonte Springs, Fl who can fix your electrical issues without a sweat. To hire electricians in Altamonte Springs, Fl, call us or send an email for a free consultation.


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A 27-year-old man confessed to raping and killing an American scientist who was in Crete for a conference, the Greek authorities said.
The alleged confession came Monday, 11 days after the 59-year-old Suzanne Eaton was declared missing on July 4 from the conference organizer – the same day the biologist was supposed to present his research to colleagues. Two amateur explorers found Eaton's body on July 8th, nearly 200 feet in a cave used as a refuge during World War II, according to police.
The authorities identified the suspect on Thursday.
Faced with the evidence, the resident of Crete Yiannis Paraskakis told the authorities that he had struck Eaton twice with his car, and brought it, unconscious, to his trunk at the abandoned shelter. She left her there after raping her, says the Crete police. Paraskakis, who comes from the city of Chania, where Eaton disappeared, was charged with murder and rape.
Prosecutors ordered the release of Paraskakis' identity in the public security interest and an investigation into other crimes that the man could have committed, police said Thursday in a press release .
Eaton died on July 2 from suffocation, based on forensic evidence and an autopsy, police said. It was discovered with injuries to both hands and broken ribs and facial bones.
The mother of two hailed from Oakland, California, but worked in Germany. She led a research group at the Max Planck Institute for Cellular and Genetic Molecular Biology in Dresden and was a professor at the Technische Universität Dresden.
"We are devastated by this senseless tragedy," the Max Planck Institute for Molecular and Genetic Cell Biology – where Eaton's husband is also a researcher – he said in a declaration last week.
Eaton was in Crete for the fourth time, in an international meeting of scientists at the Orthodox Academy of the Greek island, according to the police. He left his room at noon for his daily walk, leaving his cell phone and personal belongings behind.
Paraskakis told the authorities that he abducted Eaton – "motivated by sexual satisfaction", Crete police Eleni Papathanasiou said at a press conference on Tuesday – around noon that day as he headed for a monument called Evelpidon in the north of island.
The man said he brought Eaton into a ventilation shaft in the abandoned shelter and left his body there, blocking the opening with a wooden palette. Next to a cemetery, he cleaned his car of potential evidence, police said.
The authorities said they had collected blood samples and confiscated the machine, along with clothes and a suspect button. Paraskakis was brought to the District Attorney's Office pending forensic, clinical and toxicological examinations.
Police began searching "immediately" after Eaton's disappearance, Crete's police director Constantinos Lagoudakis told the media. An alert came out with information and images of Eaton and volunteers joined government agencies to look for the missing woman.
Eaton's husband and children went to Crete to help, a cousin said at the Associated Press. Family members have created a Facebook page to advertise the research.
The two people who eventually found Eaton were part of that growing effort, CBS News reported.
After Eaton's body was found in the cave, he said, the authorities considered its placement and from the wheel traces that led to the shelter pit that the woman had been brought there. The body has shown signs of a violent attack and potential sexual abuse.
CCTV filming, witness interviews and autopsy led the police to identify suspects and eventually enter Paraskakis.
The authorities are investigating at least seven other attacks on pedestrians that allegedly involve a car similar to the one that struck Eaton, second at the Greek state television broadcaster, the Hellenic Broadcasting Corporation.
The women in two of the previous cases reported to the police, but the cases were closed, the issuer said. Paraskakis was arrested first for arson and injured and killed animals, the statement added, although the site does not provide further details.
While the police investigated, those who knew Eaton cried a sought-after researcher and a close friend, collaborator, and family member in more than two dozen tributes filled out from the Max Planck Institute. The Institute will likely publish a more detailed obituary in the coming days, spokeswoman Christina Beck told the Washington Post.
A public service will be held in Dresden on Tuesday, Eaton's niece, Callie Broaddus, told The Post.
The collaborators of the Eaton laboratory wrote in their tribute to their colleague's love for "the big and difficult questions of science", recalling his long interdisciplinary documents.
Family members also talked about his curiosity.
Eaton's mother remembered a daughter interested in everything that surrounded her, from the day she was born, a child "not drowsy and asleep but with his head up, his eyes alert". And Eaton's son Max said his mother was "always armed with questions".
Eaton "would show interest in any subject addressed," he wrote. "Many times I discussed with her some topics that I [I] had studied at the university and, within a week, she would have been very expert in that topic like my professors".
The family members remembered not only a scientist, but also a talented pianist who played duets with her husband, an enthusiastic gardener and a black taekwondo belt. They memorized it as a fast crossword puzzle finisher and avid reader.
The tributes focus on who Eaton was rather than on the tragic circumstances of his death.
"I made a conscious decision not to allow those facts to haunt me in memory," reads the tribute of Eaton's unnamed sister. "My memory will be of pure joy and gratitude, of love and admiration for a sister in arm, a closer confidant, a strong, kind, brilliant and selfless being who has made indelible contributions to science and has added an immeasurable beauty to our lives ".


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That is a gloomy prediction Celestra !High probability she's another victim of another sex related murder.
Too many sexual deviates out there. It isn't good to dress a baby that way, not if they care about the kid...That is a gloomy prediction Celestra !
Here is something more on the right side of the tracks . I hope you will agree .I have to agree, parents are using their hids for profit.
Sickens me.
I should say I put that link as something clowny and consonant to Whoosh's frequent "Lady Gaga," but in no way I meant it to inspire any vulgar videos down this thread. So I say if a kid performer is acting like a clown, it's Ok, but provided it's not explicitly vulgar.
I certainly agree that mentally healthy humanity doesn't need "art" like that.Talking about "Lady Gaga"...... Have you seen her latest creation ? She and Beyonce travel around poisoning people. malevolence is something they should be removed from performing. Under their influence we might have a rush of homicides.


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Matthew PG Barnett1, Anthony RJ Phillips1,2., Patricia M Harris4, Garth JS Cooper1,3
1School of Biological Sciences, Faculty of Science; Departments of 2Surgery and 3Medicine, Faculty of Medical and Health Sciences, The University of Auckland. Auckland, New Zealand.
4AgResearch Limited, Grasslands Research Centre. Palmerston North, New Zealand
Received March 31st, 2008 - Accepted April 28th, 2008
Context Insufficient maternal protein intake has been postulated to cause impaired fuel metabolism and diabetes mellitus in adult mammalian progeny, but the mechanism remains unclear. Objective To investigate the effect of a maternal low protein whey-based diet during pregnancy and lactation on pancreatic function and skeletal muscle glucose metabolism in the offspring. Animals Sprague-Dawley rats: 8 mothers and 46 offspring. Design Female rats were fed throughout pregnancy and lactation with otherwisecomplete isoenergetic diets sufficient (20% whey protein; control: n=3) or insufficient (5% whey protein; low-protein: n=5) in whey protein. From weaning all offspring ate control diet. Main outcome measures Food intake and weight gain were measured for both mothers and offspring, and in vitro functional studies of endocrine pancreas and skeletal muscle were performed on offspring at 40 and 50 days of age, respectively. Results Food intake (P=0.004) and weight gain (P=0.006) were lower in low protein than control mothers during early gestation. Offspring of low protein mothers had significant lower body weight from 5 to 15 days of age, although there was no significant difference in food consumption. Glucose, arginine- and glucose/arginine-stimulated insulin secretion from perfused pancreases isolated from low protein offspring were decreased by between 55 and 65% compared with control values. Studies in skeletal muscle demonstrated no difference in insulin sensitivity between the two groups. Conclusions Dietary whey protein insufficiency in female rats during pregnancy and lactation can evoke major changes in insulin secretion in progeny, and these changes represent a persistent functional abnormality in the endocrine pancreas.
Diabetes Mellitus, Type 2; Embryonic and Fetal Development; Pancreatic Hormones
EC50: concentration of agonist required to provoke a response halfway between the baseline and maximum responses; EEU: efficiency of energy utilization
Programming [1, 2] is a process whereby poor nutrition at an early age leads to permanent changes in an organism [2]. Early adaptations to nutritional stress permanently alter the physiology and metabolism of an organ, such that these changes continue to be expressed in the absence of the original causative events [3]. In humans, such changes are postulated to be causative of later disease, including coronary heart disease [4, 5], depression [6], and the so-called "metabolic syndrome" [1], which refers to the grouping of type 2 diabetes mellitus [1, 2], cardiovascular disease [7] and hypertension [8].
Timing of the nutritional stress appears to be critical, with earlier events more likely to cause changes which persist throughout the life of the organism [9]. Therefore, foetal and early neonatal life represent developmental stages of particular interest. In rodents, it has been well established that low maternal protein intake during gestation can result in low birth weight and subsequently leads to various metabolic disturbances in adulthood, including high blood pressure, impaired glucose tolerance, insulin resistance and altered pancreatic morphology and function [10, 11, 12, 13, 14]. Additional studies are warranted to explore the relationships linking protein nutrition in early life to the postnatal development of disease in animals, as a model for the human condition [11]. Protein is important in foetal growth and pancreatic development [15], and is often scarce and expensive in communities with a high prevalence of diabetes and the metabolic syndrome [16].
A number of comparable studies have been published in which casein-based diets are used, but there is very little literature investigating a whey-based diet. Whey protein is considered to be of higher nutritional quality than casein [17].
Our hypothesis was that a decreased whey protein intake in the maternal diet (maternal low protein) during pregnancy and lactation results in a metabolic insult in the offspring during these periods sufficient to cause impaired pancreatic function and altered glucose metabolism in the offspring once adult, and that the higher quality of the whey protein (compared with casein) would be insufficient to mitigate the low-protein insult. Female rats were fed diets either sufficient or insufficient in whey protein through gestation and lactation, and pancreatic function (the secretory response to stimuli in isolated perfused pancreases) and glucose metabolism (insulin-stimulated uptake of glucose into glycogen in the isolated soleus muscle) were studied in the offspring.
Animals and Diet
Virgin female Sprague-Dawley rats were kept at 21±1ºC, subjected to a 12:12 h light:dark cycle. Diet was standard laboratory chow (18.5% crude protein, 5% fat; NRM diet 86, NRM New Zealand Ltd., Auckland, New Zealand) supplied ad libitum prior to mating. Females in oestrus were randomly selected and mated with one of three males. Dams were assigned such that offspring of each male would be represented in both experimental diet groups, to ensure any observed results were not due to a simple genetic effect. A positive pregnancy was determined by the appearance of a vaginal plug (defined as day 1 of gestation), at which stage the rats were transferred to individual cages. Throughout gestation and lactation rats were fed isoenergetic semi-synthetic diets containing either 20% whey protein (control), or 5% whey protein (low-protein) (Table 1). Diets were prepared in the laboratory from commercially available ingredients, and compositional analyses were performed on the diets (Table 1). A control diet of similar composition has been previously reported [18]. During gestation, animals were not fed ad libitum. Instead, the amount fed daily to each pregnant rat was calculated from the body weight of the rat and the particular day of gestation. This calculation was based on food intake data from a previous feeding trial for mothers fed standard laboratory chow (data not shown), with the food restriction being applied to prevent low protein mothers consuming more than control mothers during gestation, as has been previously observed [19]. All animals had free access to water.
Following spontaneous delivery of pups (day 22-23 of gestation), mothers were maintained on the same diet as had been supplied during gestation, without restriction on intake. No restriction was applied during lactation because observations suggest that higher food intake in low protein mothers does not occur during this period [19, 20]. At 21 days of age, pups from all litters were weaned onto the control diet, and this was supplied ad libitum for the remainder of the trial.
Dietary Analysis
Nitrogen determination was by the instrumental combustion method (Nitrogen Analyser Series 1500, Carlo Erba, Serono, Italy), lipid determination by the Soxhlet method [21], and carbohydrate determination by an enzymatic and spectrophotometric method [22, 23]. Acid stable amino acids were analyzed by acid hydrolysis, sulfur amino acids by performic acid oxidation followed by acid hydrolysis [24]. Protein was calculated from the nitrogen content (Nx6.38) [25, 26], and total energy as net metabolisable energy was calculated from the individual components as has previously been described [27].
Maternal food intake and body weight were measured daily from the first day of gestation until 21 days post-partum (weaning). Gestation was divided into two equal periods (equivalent to the first and second trimesters of human pregnancy [28, 29]) in order to determine if any observations in the offspring were due to differences in maternal intake or body weight during a specific part of gestation.
Offspring body weight was measured daily from birth until 50 days of age, while food intake (determined as mean intake per rat within a litter) was measured daily from weaning until 50 days of age. Feeding efficiency for both mothers and offspring was calculated as the efficiency of energy utilization (EEU; total weight gain / total energy intake, g/kJ) as previously reported [30].
Isolation and Perfusion of the Pancreas
Non-fasted offspring (both male and female) were used as pancreas donors (age at experimentation 42±2 days). A total of 5 (2 litters) and 8 (3 litters) animals were used from the control and low protein groups, respectively. After initial anaesthesia by halothane induction (5% plus 2 L/min O2) an intra-peritonal injection of sodium phenobarbitone (Nembutal®, Sigma, St Louis, MO, USA) 50 mg/kg body weight) was performed and animals maintained on oxygen during subsequent removal of the pancreas. Pancreases were prepared according to the method of Grodsky and Fanska [31], with minor modifications. Briefly, the pancreas, stomach, and duodenal remnant were isolated en bloc, the aorta and portal vein cannulated, and the preparation transferred to a perfusion chamber. Perfusion was performed simultaneously through the coeliac and superior mesenteric arteries (flow rate 1.2 mL/min) and effluent was collected without recycling from the cannulated portal vein. The perfusion medium was modified Krebs- Henseleit buffer (final concentrations: NaCl, 112.8 mmol/L; KCl, 4.4 mmol/L; KH2PO4, 1.5 mmol/L; MgSO4, 1.2 mmol/L; CaCl2, 2.3 mmol/L; NaHCO3, 2.93 mmol/L; D-glucose, 3 mmol/L), supplemented with dextran (4% (w/v), molar weight 71,400, industrial grade; Sigma, St Louis, MO, USA) and albumin (0.5% (w/v), bovine fraction V; A-6793, Sigma, St Louis, MO, USA). Buffer was sterile filtered (0.2 μm; Vacucap, Gelman, Ann Arbor, MI, USA) before equilibration against a gas phase of 95% O2/5% CO2 (BOC Gases NZ Ltd., Auckland, New Zealand), and had a final pH of 7.4 at 37°C.
Stimulation of the Pancreas and Sample Collection
Organs were equilibrated for 20 min. For 90 min thereafter portal vein effluent fractions were collected at 1 min intervals and stored on ice. Each gland was stimulated during the experiment by means of side arm infusions (0.05 mL/min) with either glucose (final concentration 21.7 mmol/L), arginine (final concentration 10.85 mmol/L), or a mixture of the two. Both glucose and lactate were measured simultaneously on every second sample with a glucose/lactate analyser (YSI 2300 Stat Plus, Yellow Springs Instrument Co., Yellow Springs, OH, USA); glucose to ensure that the final perfusate concentration was as expected, and lactate to confirm pancreas viability [32]. Samples were stored at -80°C until analysis for insulin and amylin content.
Insulin Radioimmunoassay
Perfusate insulin concentration was determined in duplicate at each point using a double antibody RIA. Briefly, antibodies were raised against bovine insulin in guinea pigs. Radiolabelled tracer (125I-insulin) was prepared by iodination of bovine insulin using the chloramine-T method [33], and purified by gel filtration chromatography. The standard curve was generated using serial dilutions of soluble human insulin (Actrapid, Novo Nordisk, Copenhagen, Denmark) as a standard. The minimum detectable concentration was 16 pmol/L, the linear range 140-1,800 pmol/L, and the mean within-assay coefficient of variation 19% at the EC50.
Duplicate 50 μL samples of standards or unknowns were incubated with 200 μL of antiserum (diluted 1/100,000 in RIA buffer (0.05 mol/L phosphate buffer, 0.1% (v/v) sodium azide, 0.5% (w/v) bovine serum albumin, pH 7.4)). After 5 h at room temperature, 100 μL of tracer (diluted in insulin RIA buffer to give a reading of approximately 9,000 cpm/100 μL) was added and incubated for 48 h. One hundred μL of goat anti-guinea pig IgG (diluted 5/100 (v/v) in RIA buffer), and 100 μL of normal guinea pig serum (1/100 (v/v) in RIA buffer) were added and incubated for 1 h at room temperature. One mL of polyethylene glycol 6000 (6% (w/v) in 0.05 mol/L phosphate buffer, pH 7.4) was added, followed by further incubation for 90 min at 4°C. Tubes were spun at 1500 g for 30 min, decanted and counted for 1 min in a gamma counter (WizardTM, Wallac, Turku, Finland).
Amylin Radioimmunoassay
Perfusate amylin concentration was measured in duplicate at each point using an in-house RIA (M. Stokjovic, unpublished results). Amylin antibodies were raised in rabbits using synthetic rat amylin (Bachem AG, Bubendorf, Switzerland). Radiolabelled tracer (125I-rat amylin) was prepared by iodination of purified synthetic rat amylin (Bachem AG, Bubendorf, Switzerland) using the chloramine-T method [33], and purified by gel filtration chromatography. The assay was standardised using lyophilised rat amylin standards (Peninsula Laboratories, Belmont, CA, USA). The minimum detectable concentration was 25 pmol/L, the linear range 25-250 pmol/L, and the mean within-assay coefficient of variation 22% at the EC50.
Duplicate 100 μL samples of standards or unknowns were incubated with 100 μL of antiserum (diluted 1/100,000 in RIA buffer (0.05 M phosphate buffer, 3.1 mmol/L sodium azide, 0.25% (w/v) bovine serum albumin, 0.1% (v/v) Triton X-100, 25 mmol/L EDTA, 1% aprotonin, pH 7.4). After 24 h at 4°C, 100 μL of tracer (diluted in amylin RIA buffer to give a reading of approximately 9,000 cpm/100 μL) was added and incubated for 48 h (4°C). One hundred μL of goat antirabbit IgG diluted 1/15 (v/v) in buffer and 100 μL of normal rabbit serum diluted 1/100 (v/v) were added and incubated for 1 h at room temperature. Polyethylene glycol 6000 (0.5 mL, 8% (w/v) in 0.05 mol/L phosphate buffer) was then added followed by a further incubation for 90 min at 4°C. Samples werem centrifuged at 1,500 g at 4°C for 30 min, decanted and counted for 1 min in a gamma counter. Data analysis for both assays used MulticalcTM software version 2.0 (Wallac, Turku, Finland).
Insulin-Stimulated Uptake of Glucose into Muscle Glycogen
Male and female offspring (13 animals from three litters for control group, and 20 animals from four litters for low protein group; age: 50±2 days) were starved overnight (16-18 h) with free access to water. An intra-peritoneal injection of sodium phenobarbitone (80 mg/kg body weight; Nembutal®, Sigma, St Louis, MO, USA) was administered, and the animals sacrificed by cervical dislocation once the anaesthetic had taken effect. The soleus muscle was removed from each leg under Krebs-Henseleit buffer (final concentrations (mmol/L): NaCl, 118.5 mmol/L; KCl, 4.75 mmol/L; MgSO4, 1.18 mmol/L; NaHCO3, 24.8 mmol/L; KH2PO4, 1.18 mmol/L; CaCl2, 2.54 mmol/L; Dglucose, 10 mmol/L) saturated with 95% O2/5% CO2. Each soleus muscle was then split longitudinally into two or three strips, depending on the muscle diameter. After 20 min equilibration in a separate container of 95% O2/5% CO2-saturated Krebs-Henseleit buffer, up to five strips were transferred to a 50 mL Erlenmeyer flask in a 30°C shaking waterbath. Each flask contained 10 mL of normal Dulbecco's Modified Eagle Medium with one of the following concentrations of insulin: 0, 0.71, 2.37, 7.1, 23.7, 71.0, 237.0 nM. Exactly 2 min after the transfer of muscle strips to the flask, 10 μL of a 1.85 MBq/mL solution of U-14C-D-glucose (American Radiolabeled Chemicals Inc., St Louis, MO, USA) were added, and the flask incubated for a further 2 h at 30°C under a constant stream of 95% O2/5% CO2. Each muscle strip was then snap-frozen in liquid nitrogen and freeze-dried for a minimum of 24 h.
Glycogen Extraction from soleus Muscle and Scintillation Counting
Freeze-dried muscle was digested in 250 μL of 60% (w/v) KOH for 45 min at 70°C and the glycogen precipitated by addition of 750 μL cold 95% ethanol. Samples were kept at - 20°C overnight then centrifuged (10,000 g, 15 min, 4°C) and the supernatant aspirated. The pellet was washed in 750 μL cold 95% ethanol, centrifuged and the supernatant aspirated. The wash step was repeated once more and the glycogen pellet dried at 60°C for 2 h. Glycogen was redissolved in 200 μL deionized water and transferred to a scintillation tube. StarScint® (Canberra Packard, Pangbourne, Berkshire, UK) scintillation fluid (1.8 mL) was added and the sample counted for 5 min (LS 3801 betacounter, Beckman Coulter Inc., Fullerton, CA, USA).
Blood was collected into lithium heparin vacutainers immediately post mortem from the vena cava of animals sacrificed for the removal of soleus muscle. Plasma was separated from the red cells and stored at - 85°C until measurements were performed. Glucose and lactate concentration (control: 13 animals from three litters; low protein: 13 animals from four litters) was measured using a glucose/lactate analyzer (Yellow Springs Instrument Corp., Yellow Springs, OH, USA), while cholesterol and triacylglycerol (control: 9 animals from three litters; low protein: 3 animals from four litters) were determined using a Cobas Mira analyzer (Roche, Basel, Switzerland).
All experiments performed were approved by the University of Auckland Animal Ethics Committee.
All statistical analyses were performed using GenStat® for Windows 5th edition or 6th edition (GenStat® Release 4.22, PC/Windows XP, Lawes Agricultural Trust, Rothamsted Research, Harpenden, Hertfordshire, United Kingdom, Version 18.104.22.168). Unless otherwise stated, analysis was a linear mixed model residual maximum likelihood (REML), with diet being the fixed effect and litter the random effect. Unless otherwise stated, data are presented as the mean value for each of the control and low protein groups, with the mean and standard error of the difference (difference±SE) for the comparison between the two groups. Graphs show mean values and standard deviations (SD).
For the insulin-stimulated uptake of glucose into glycogen, a four-parameter logistic curve was fitted to the data with the equation:
y = ( a - b ) / ( 1 + (insulin/c) d ) + b
where the parameters were defined as follows:
a is the asymptote as x tends to 0 for all d greater than 0 (i.e. the minimum value);
b is the asymptote as x diverges (i.e. the maximum value);
c is the predicted response midway between the asymptotes (i.e. the effective insulin concentration at which the half-maximal response occurs (EC50));
d is a function of the rate of change (or slope) of the fitted curve at the point of inflection.
Because the maternal diet was altered, the experimental unit in all cases was the litter; thus, the effective number of observations for each experiment was determined by the number of litters rather than the number of individual offspring. A two-tailed probability lower than 0.05 indicated a significant difference between treatments.
Maternal Food Intake, Body Weight and Feeding Efficiency
Food intake in the low protein group was significantly lower than the control group during the first half of gestation (control: 248 g; low protein: 219 g; difference±SE: -29±11 g; P=0.004) (Figure 1a). As with food intake, energy intake (net metabolisable energy) during the first period of gestation was significantly lower in the low protein mothers compared to those in the control group (control: 4,197 kJ; low protein: 3,708 kJ; difference±SE: -489±186 kJ; P=0.008).
Figure 1. Food intake and body weight of female Sprague-Dawley rats fed through gestation and lactation with diets varying in protein content. Data represent mean values±SD for daily food intake (a.), and for change in body weight from the first day of gestation (b.). Mothers were fed iso-energetic diets (prepared in our laboratory) containing either 20% (control: filled circle) or 5% (low-protein: open circle) whey protein through gestation and lactation. The numbers within brackets above each figure represent the trimester of human gestation to which each indicated period is equivalent. The total number of observations was 5 and 6 mothers for the control and low-protein groups, respectively. P values indicate the trimester equivalents during which there was a significant difference between the two diet groups.
Total weight gain of the mothers through gestation in the low protein group was lower than that in the control group (control: 117 g; low protein: 78 g; difference±SE: -39±9 g; P<0.001), primarily due to a difference during the first half of gestation (control: 40 g; low protein: 10 g; difference±SE: -30±11 g; P=0.006) (Figure 1b). Weight change showed no difference during the second half of gestation (control: 77 g; low protein: 68 g; difference±SE: -9±8 g; P=0.285) or through lactation (control: -23 g; low protein: -18 g; difference±SE: 5±15 g; P=0.766).
Feeding efficiency (EEU: efficiency of energy utilization) was significantly lower in the low protein group than the control group during gestation (control: 0.015 g/kJ; low protein: 0.011 g/kJ; difference±SE: -0.004±0.001 g/kJ; P<0.001) and lactation (control: 0.015 g/kJ; low protein: 0.008 g/kJ; difference±SE: - 0.007±0.002 g/kJ; P<0.001). As with weight gain and food intake, the overall difference during gestation was mainly due to a significant difference during the first half of gestation (control: 0.009 g/kJ; low protein: 0.003 g/kJ; difference±SE: -0.006±0.003 g/kJ; P=0.016). The calculation for lactation included the weight gain by offspring during this period, as offspring growth was dependent on maternal food intake.
Offspring Food Intake and Body Weight
There was a small but significant (ranging from P=0.003 to P=0.049 for the comparison on each day) difference in body weight of the offspring between day 5 and day 15 inclusive, with the low protein offspring weighing less than those in the control group (Figure 2b). There was no significant difference with respect to birth weight (control: 5.4 g; low protein: 5.4 g; difference±SE: 0.0±0.4 g; P=0.990), total weight gain (control: 97 g; low protein: 92 g; difference±SE: -5±11 g; P=0.641), or rate of weight gain either preweaning (control: 1.2 g/day; low protein: 1.0 g/day; difference±SE: -0.2±0.2 g/day; P=0.257) or post-weaning (control: 2.7 g/day; low protein: 2.8 g/day; difference±SE: 0.1±0.8 g/day; P=0.760) between the two groups (Figure 2b). There was no difference between the two groups in terms of offspring food intake (Figure 2a; control: 225 g; low protein: 203 g; difference±SE: -22±19 g;P=0.257).
Figure 2. The effect of protein level in the maternal diet during gestation and lactation on food intake and body weight of offspring. Data represent mean values±SD for daily food intake (a.) and body weight (b.) of offspring of mothers fed isoenergetic diets (prepared in-house) containing either 20% (control: filled circle) or 5% (low-protein: open circle) whey protein through gestation and lactation. All offspring were weaned (indicated by the dashed vertical line) onto diet control. The total number of litters was 5 for control and 6 for low protein groups. There was no significant difference in pre-weaning weight gain, or post-weaning weight gain or food intake, between the two diet groups.
Table 2 shows the effect of maternal diet on the secretion of insulin from the isolated perfused pancreas. Total insulin secretion, mean basal insulin secretion and secretion in response to each of the three stimulations (glucose, arginine, and glucose plus arginine) were significantly reduced in low protein offspring. In the case of the response to glucose, both first-phase (control: 27 pmol; low protein: 9 pmol; difference±SE: -18±5 pmol; P<0.001) and second-phase (control: 30 pmol; low protein: 11 pmol; difference±SE: - 19±11 pmol; P=0.001) secretion were reduced in the low protein group, while in the response to arginine, only the second-phase secretion was reduced (control: 5.3 pmol; low protein: 2.2 pmol; difference±SE: -3.1±1.4 pmol; P=0.025). In the case of the stimulation by the combined glucose plus arginine solution, the low protein offspring showed a loss of first-phase insulin secretion, both as a significant decrease compared to the control group (control: 34 pmol; low protein: 11 pmol; difference±SE: -23±7 pmol; P<0.001) and first-phase as a percentage of total secretion resulting from this stimulus (control: 40%; low protein: 30%; difference±SE: - 10±2%; P<0.001).
Neither amylin secretion, nor the ratio of amylin/insulin, showed any significant difference between the two diet groups (data not shown).
Insulin-Stimulated Uptake of Glucose into Muscle Glycogen
Glucose uptake into soleus muscle glycogen was not significantly different in low protein animals compared to those in the control group at any point of the dose-response curve (Figure 3). There was no significant difference between the two groups with respect to minimum (P=0.459), maximum (P=0.842), EC50 (P=0.302) or slope (P=0.362) as determined by four-parameter logistic curve fitting.
Figure 3. Incorporation of 14C-glucose into soleus muscle glycogen of offspring of Sprague-Dawley females fed diets differing in protein content during gestation and lactation. Rates of 14C glucose uptake into glycogen were determined in rat soleus muscle isolated from the offspring of mothers fed diets sufficient (20% whey protein; control: filled circle) or insufficient (5% whey protein; low-protein: open circle) in protein. Data represent the mean value±SD for 13 animals from three litters for control group, and 20 animals from four litters for low-protein group. There was no significant difference between the two groups at any insulin concentration.
There was no difference between offspring of the two diet groups in terms of plasma levels of glucose (control: 4.3 mmol/L; low protein 6.7 mmol/L; difference±SE: 2.4±2.0 mmol/L; P=0.241), lactate (control: 4.1 mmol/L; low protein: 4.9 mmol/L; difference±SE: 0.8±0.8 mmol/L; P=0.301) or cholesterol (control: 2.1 mmol/L; low protein: 2.2 mmol/L; difference±SE: 0.1±2.3 mmol/L; P=0.808), but low protein offspring had a significantly lower plasma triacylglycerol concentration (control: 0.93 mmol/L; low protein: 0.50 mmol/L; difference±SE: -0.43±0.13 mmol/L; P=0.001) than control offspring.
In general, the pattern of food intake and body weight observed in the low protein mothers was as has been reported. A sharp decline in intake over the last days of gestation in the low protein group [34] was reflected in the slowing of weight gain towards the end of gestation [19], while the low intake during lactation [19, 35] caused a loss of weight through lactation [19]. Food intake in the low protein group during gestation was unusual in that it tended to be less than in the control group; therefore, there was no evidence of compensatory feeding [19, 20]; the restriction on intake imposed during gestation seems to have been unnecessary in this case. In fact, the lower maternal food (and therefore energy) intake in the low protein group means that the offspring of these dams were exposed to both caloric and protein restriction during early gestation. Although the caloric restriction was much less than the protein restriction, we cannot exclude the possibility that this has influenced the outcomes reported here. In future studies we would employ a more strict dietary regime such that caloric intake was the same for both diet groups.
The observed food intake of the control group mothers was unusual. While the decline in food intake for control rats before parturition (similar to that seen in the low protein group) is routinely observed, the magnitude of this decline, and the low intake throughout lactation, are at variance with our own studies using standard laboratory chow (data not shown), and previously reported data [36, 37]. This suggests that the protein content of the diet is not the only factor controlling food intake. The pattern of maternal body weight in the control group reflected the pattern of food intake, with a slowing in growth rate at the end of gestation and an average weight loss of over 20 g per dam through lactation, neither of which have been observed in previous studies using a similar diet [20, 36].
Offspring in both diet groups (all of which were weaned onto control diet) consumed similar amounts of food through the duration of the trial, but this amount was significantly less than we observed in animals fed standard laboratory chow (data not shown). Body weight in both groups was similar, with the slightly lower weight observed in the low protein group from day 5 to day 15 presumably a reflection of the tendency to lower food consumption in the low protein mothers through lactation. In both cases, body weight was significantly less than previously reported values [20].
In the isolated perfused pancreas, low protein offspring showed evidence of impaired pancreatic function in response to the main insulin secretagogue (glucose) and a membrane depolarizing agent (arginine). In this particular experiment, there was a significant difference in each of the stimulations applied to the pancreas. The loss of glucose-induced insulin secretion is in agreement with previous studies showing a decreased insulin secretion both in vivo and using an in vitro isolated islet system [38]. There was also an impairment of first-phase insulin secretion in response to a combined glucose/arginine stimulation. A loss of first phase glucose-induced insulin secretion is a feature of the early stages of type 2 diabetes [39, 40] and could be due to either exhaustion of the labile pool of insulin granules or a time-dependent inhibition of the secretion response [39]. There is evidence that the islet size, proliferative capacity and vascularization [15], as well as the amount of beta cells within each islet and the percentage of islet tissue and beta cells within the pancreas [41] are all reduced in the pancreases of offspring of rat dams fed a low protein diet during gestation [15, 41] and/or lactation [41]. The lower insulin secretion observed in low protein offspring may be due to one or more of these factors.
Previous studies show an inhibitory effect by amylin on insulin secretion [42, 43] at a concentration as low as 75 pmol/L, and it was for this reason that amylin secretion was measured. Secretion levels higher than 100 pmol/L were observed in low protein offspring during the basal phase, although there was considerable variation between animals. Because of this variability, it is unclear whether or not amylin is impairing the insulin secretion from low protein offspring. Additional studies employing, for example, a specific amylin antagonist such as the amylin8-37 molecule (a truncated peptide of native rat amylin in which the first seven amino acids are absent) [44] would be helpful in clarifying this.
A number of studies have been performed which suggest that an increase in peripheral insulin sensitivity in the offspring of proteinrestricted rats may be an adaptive response to a reduction of insulin secretion. These include both low protein [45] and protein-free [46] diets during early lactation only, and low protein during both gestation and lactation [38]. However, in the current study, there was no evidence of an increase in insulin sensitivity in the low protein offspring. The reasons for this observation are unclear; it would be expected that decreased insulin secretion would lead to a higher plasma glucose concentration in the absence of any change in insulin sensitivity. No such change in plasma glucose was observed, although the insulin sensitivity of tissues such as liver and adipose was not measured in the experiments reported here. Further studies incorporating such measurements are required to clarify this issue.
Finally, the observation that fasting plasma triacylglycerol levels were lower in low protein offspring is in agreement with previous studies [47], suggesting that in this instance a maternal low protein diet during gestation and lactation may program risk for diabetes, but not for vascular disease, as may be the case for early human undernutrition. The possibility cannot be excluded that changes relevant to vascular disease may occur after 50 days of age, the point at which these experiments ended.
Overall, these results show a decrease in pancreatic insulin secretion in the offspring of female rats fed a low whey protein diet during gestation and lactation; such a decrease is associated with the early development of type 2 diabetes in humans and suggestive of an increased susceptibility to the development of this disease in adult life, as has been observed in similar studies using casein as a protein source. This observation is in accordance with the hypothesis that the fetal/neonatal environment plays a role in the programming of type 2 diabetes.
The authors wish to thank Jenny Rains for assistance with animal handling and timed matings, Dr. Christina Buchanan for assistance with perfused pancreas and radioimmunoassay, Dr Bernard Choong and Dr Mirjana Stokjovic for iodination of hormones, Harold Henderson for assistance with statistical analyses and fitting of the four-parameter logistic model, Dr Nicole Roy for constructive criticism of the manuscript, and Denise Martin for assistance with preparation of the manuscript. The current affiliation of Matthew Barnett is: Food, Metabolism & Microbiology, Food & Textiles Group, AgResearch Limited, Grasslands Research Centre, Palmerston North, New Zealand. The current affiliation of Patricia Harris is: The Foundation for Research, Science and Technology, 15-17 Murphy Street, PO Box 12-240, Wellington, New Zealand. This research was funded by Endocore Research Trust. Matthew Barnett was supported by Doctoral Scholarships from The Agricultural and Marketing Research and Development Trust (AGMARDT) of New Zealand and The University of Auckland, and by Endocore Research Trust
All authors confirm that there are no potential conflicts of interest.


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The roots of European witchcraft and magic lie in Hebrew and other ancient Near Eastern cultures and in the Celtic, Nordic, and Germanic traditions of the Continent. For two millennia, European folklore and ritual have been imbued with the belief in the supernatural, yielding a rich trove of histories and images.
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I'm sure it's pure coincidence that Louisiana's two favorite contact sports — politics and football — align closely on the calendar. Anticipation runs high among New Orleans Saints and LSU fans, and next week similarly marks the official kickoff of the political season.
Qualifying begins Tuesday, Aug. 6 and closes Thursday, Aug. 8 for statewide offices, legislative seats and local offices across the state. Just as in the other contact sport, we're bound to see some surprises in the political arena even as the season just begins.
The governor's race always draws the most interest, and often the most candidates. Democratic incumbent John Bel Edwards consistently leads in all polls, but his two main GOP opponents — Congressman Ralph Abraham of Alto (in northeast Louisiana) and businessman Eddie Rispone of Baton Rouge — are determined to spend millions between them to make it a close race. It'll be interesting to see if any other big-name candidates jump into this race at the last minute. I make no predictions.
The down-ballot contests always seem to get short shrift, even those for statewide offices. Truth is, there hasn't been much noise about those races, not even among challengers. The one exception appears to be the race for insurance commissioner. Incumbent Jim Donelon will seek a fourth full term after having been appointed in 2006. His only announced opponent is insurance executive Tim Temple of DeRidder, who promises to give Donelon a run for his money.
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text: Original Articles: GastroenterologyDexamethasone Changes the Composition of Insulin-like Growth Factor Binding Proteins in the Newborn Mouse IleumGordon, Phillip V.*; Moats-Staats, Billie M.†; Stiles, Alan D.†; Price, Wayne A.†Author Information *Division of Neonatology, Department of Pediatrics, University of Virginia Health Sciences, Charlottesville, Virginia, U.S.A., and †Division of Neonatal–Perinatal Medicine, Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A. Received October 29, 2001; accepted May 30, 2002. Supported by grants no. HL1917l-18 SCOR (to A.D.S. and B.M.M.S.) and CHRCDA HD01421 (to P.V.G) from the National Institutes of Health, Bethesda, MD, and by start up funds from the Department of Pediatrics, University of Virginia Health Sciences, Charlottesville, VA. Address correspondence and reprint requests to Dr. Phillip V. Gordon, Division of Neonatology, Department of Pediatrics, University of Virginia Health Sciences, Charlottesville, VA 22908–0386 (e-mail: firstname.lastname@example.com). Journal of Pediatric Gastroenterology and Nutrition: October 2002 - Volume 35 - Issue 4 - p 532-538 Free Abstract Background Early postnatal glucocorticoid exposure accelerates the maturation of the bowel mucosa but results in bowel wall thinning in the newborn mouse ileum and increases the risk of focal ileal perforation in extremely premature infants. We have previously demonstrated a redistribution of insulin-like growth factor-I (IGF-I) from the submucosa in control animals to the distal villi of those treated with early postnatal dexamethasone, implicating IGF-I as an important mediator of dexamethasone's capacity to alter tissue growth. To investigate the possibility that IGF binding proteins (IGFBPs) might contribute to this process, we characterized the localization and abundance of IGFBP peptides and mRNAs in the same model. Methods Newborn mice received daily intraperitoneal injections of dexamethasone (l μg/g) or phosphate-buffered saline and then were euthanized on day 3 of life. Their ileums were harvested and prepared for microscopy. Tissue sections of ileum from both treatment conditions were processed in parallel for immunolocalization of each of the six IGFBP peptides and for in situ hybridization of each of the six IGFBP transcripts. Results Transcripts for IGFBP-1, -2, and -3 were highly abundant and ubiquitous the ileal mucosa, whereas transcripts for IGFBP-4, -5, and -6 were less abundant in epithelial cells. There were no differences in abundance between control and dexamethasone-treated ileum with regard to mRNA localization or abundance for IGFBP-1, -2, -3, and -6. In contrast, mRNA transcripts for IGFBP-4 and -5 were modestly increased with dexamethasone treatment (although only IGFBP-4 was significant). Strikingly different patterns of IGFBP immunolocalization were observed between control and dexamethasone-treated animals. IGFBP-1, -2, -3, and -5 were not detected in control ileum, whereas IGFBP-4 and -6 were both present in the mucosa. In contrast, dexamethasone treatment resulted in dramatic mucosal increases in IGFBP-2, -3, -4, and -5, paralleling the changing distribution of IGF-I that we previously reported. Conclusion Taken together, these findings further implicate the IGF system as an important participant in dexamethasone-induced maturation in the newborn mouse ileum. Insulin-like growth factors (IGFs) are important mediators of bowel growth and maturation (1–3). We have previously implicated IGF-I as a mediator of dexamethasone effect in the newborn mouse ileum (4). This model was originally designed to mimic a clinical disorder observed in extremely low-birth-weight infants, where early postnatal dexamethasone treatment results in an increased incidence in focal small bowel perforation within the ileum (5–7). Both dexamethasone-treated newborn mice and dexamethasone-treated extremely low-birth-weight infants who acquire these ileal-specific perforations demonstrate robust mucosal growth concomitant with a thinning of the bowel wall. This altered growth pattern parallels the redistribution of IGF-I, suggesting it as an important intermediate of dexamethasone effect (Fig. 1) (8).FIG. 1.: Schematic drawing of insulin-like growth factor-I (IGF-I) redistribution from the submucosa and lamina propria to the distal villus in the newborn ileum of mice who received daily intraperitoneal injections of 1 μg/g dexamethasone. This therapeutic regimen has been shown to result in precocious maturation of the mucosa (4).Although steroids are known to affect tissue growth and maturation in many neonatal organs, the mechanism(s) by which glucocorticoids affect the IGF system remain poorly characterized. Several mechanisms may be involved in dexamethasone-mediated IGF-I redistribution, including regional changes in 1) IGF-1 synthesis, 2) IGF-I uptake and degradation, or 3) IGF binding protein (IGFBP) abundance. IGFBPs bind IGF-1 with variable affinity, and their individual effects can either inhibit or augment the availability of IGF-I to bind to its receptor (1). In addition, individual IGFBPs also have separate affinities for different extracellular matrix constituents, and these interactions may be important for region-specific localization of individual IGFBPs. We hypothesized that dexamethasone alters the distribution of IGF-I in the ileum by changing the mucosal composition of IGFBPs. To test this hypothesis, we assayed for changes in the abundance and distribution of all six IGFBPs and their mRNAs within the tissues of the newborn mouse ileum after dexamethasone treatment. MATERIALS AND METHODS Dexamethasone Administration and Tissue Collection The animal protocols in this study were reviewed and approved by the institutional animal care and use committee at the University of North Carolina. Newborn C57 littermates from six litters were pooled and then equally distributed between the six mothers. Three litters received daily intraperitoneal injections of dexamethasone (1 μg/g), while three litters received saline beginning on the day of birth and repeated each 24 hours for three doses. This dose and length of therapy approximates the original daily dose used in a clinical trial where 17% of dexamethasone-treated infants in the first phase of the study developed focal small bowel perforation (this prompted the safety review committee to lower the dexamethasone dose, and there was a significant decrease in perforations over the remainder of the study) (6). We chose to use intraperitoneal injections rather than subcutaneous or intramuscular injections because this best approximated intravenous delivery, which was not feasible in this animal model. Six litters of mice were used. Pups were killed after 72 hours of treatment (in the satiated state), and the ileum was immediately removed, fixed in 10% buffered paraformaldehe (Fisher Scientific, Pittsburgh, PA), and embedded in paraffin. Probe Labeling The cDNAs used in this study have been previously described and include rat IGFBP-l (a gift from Dr. L. Murphy, University of Mannitoba, Winnipeg, Manitoba, Canada), rat IGFBP-2, rat IGFBP-3, rat IGFBP-4, rat IGFBP-5, and rat IGFBP-6 (9). Riboprobes for use in the in situ hybridization analyses were generated from rat IGFBP-1–6 cDNAs by in vitro transcription using fluorescein-isothiocyanate conjugated–UTP (Boerhinger Mannheim Corp., Indianapolis, IN) and T7 DNA-dependent RNA polymerase (antisense probes) or T3 DNA-dependent polymerase (sense strand probes) (Stratagene, La Jolla, CA) (9). In Situ Hybridization In situ hybridization was performed as previously described with some modification (9,10). The ileums from 3-day-old mice were dissected immediately on euthanasia and placed directly in 10% buffered paraformaldehyde (Fisher Scientific) at 4°C overnight before being paraffin embedded and sectioned for microscopy. Sections from dexamethasone and control mice were paired and processed on the same slide for the purpose of comparison. Sections were deparaffinized in xylene, then hydrated in decreasing ethanol concentrations to distilled water, digested in proteinase K (10 mg/ml in 100 mmol/L Tris-HC1, pH 8.0, Boehringer Mannheim) for 15 minutes at 37°C, then dehydrated in increasing ethanol concentrations and air dried before hybridization. Hybridization solution (50% deionized formamide, 0.25 mg/ml yeast transfer RNA in 4X SSC [20X SSC = 3 mol/L NaCl, 0.3 mol/L sodium citrate, pH 6.0], 5 ng fluorescein-isothiocyanate conjugated UTP-labeled probe/section) was applied to each section and covered with a glass cover slip. A sense strand RNA probe for each of the IGFBPs was prepared as previously described (18). Sections were incubated in a humidified chamber at 60°C for 16 to 18 hours. Coverslips were then removed in IX SSC, and sections washed in IX SSC for 60 minutes at 68°C. Slides were washed 4 times at 60°C in IX SSC, then twice in buffer 1 (150 mmol/L NaCl, l00 mmol/L Tris-HC1, pH 7.5). Sections were then soaked in buffer 1 containing 1% bovine serum albumin (Boerhinger Mannheim Corp.) for 30 minutes at 25°C. Anti–fluorescein-isothiocyanate conjugated horse radish peroxidase (1.5 U/mL; Boerhinger Mannheim Corp.) was added in the same buffer and incubated for 2 hours. This was then removed by aspiration, and the slides were washed four times with buffer 1 at 25°C. Biotinyl tyramine (0.007 mmol/L) plus 0.003% H2O2 in buffer 1 was added, and the sections were incubated for a further 7 minutes followed by addition of avidin-biotin-peroxidase (ABC-HRP; Vector Labs, Burlingame, CA) and incubation for 30 minutes at 25°C. Sections were then washed and detection performed in substrate solution (0.05% 3´-3-diaminobenzidine, 0.003% H2O2 in 0.1 mol/L sodium acetate, pH 6.0), which was applied for 5 minutes to allow signal detection. After detection, sections were stained with hematoxylin, coverslipped, and digitized using a Spot Jr. digital camera (Diagnostics, Inc., Sterling Heights, MI). Specificity controls for in situ hybridization were performed by two methods. First, in situ hybridization was performed in parallel on adjacent sections, but RNA probe was excluded to determine the level of background staining. Second, in situ hybridization was performed as described except that adjacent sections of tissue were pretreated with a cocktail of three RNases (RNase A 1 mg/mL, Roche, Indianapolis, IN; Tl l:50 Ambion's RPA III kit, Ambion, Austin, TX; RNase H 10 U/mL, Promega, Madison, WI) for 1 hour at room temperature to test for nonspecific hybridization. Immunocytochemistry To allow direct comparisons, dexamethasone-treated and untreated tissues were processed on the same slide as previously described (4). Sections (4 μm) were deparaffinized in xylene and hydrated in descending ethanol concentrations to distilled water. Antigen retrieval was performed by incubating slides in 0.1 mol/L citric acid, pH 8.0, for 10 minutes at 100°C, followed by cooling to 25°C and washing three times in distilled H2O. Endogenous peroxidase activity was quenched in 3% H2O2 in 60% methanol for 15 minutes, followed by three washes of distilled water. Blocking solution (1% wt/vol bovine serum albumin in TBS [0.05 mol/L Tris-HCL, 0.138 mol/L NaCl, 0.0027 mol/L KCl, pH 8.0]) was applied for 1 hour at 25°C, followed by incubation with one of six antibodies, rabbit anti-human IGFBP-l (a gift from Dr. David Clemmons, University of North Carolina at Chapel Hill) (11,12), goat anti-human IGFBP-2–5, (Santa Cruz Biotechnology Inc., Santa Cruz, CA), and rabbit anti-human IGFBP-6 (a gift from Dr. Nicolas Ling) (13) for 16 to 18 hours at 4°C in a humidified chamber. The primary antibody solution was aspirated, and the slides were washed three times at 25°C in TBS. Biotinylated anti-mouse antibody (Vector Labs) or biotinylated anti-rabbit antibody (Jackson Labs, West Grove, PA) was prepared per the manufacturer's instructions and applied for 60 minutes at 25°C, followed by three TBS washes as above. ABC-HRP (Vector Labs) was prepared according to the manufacturer's instructions, applied for 30 minutes, and then washed as above. The peroxidase substrate solution was then applied to each section for 5 minutes. The reaction was terminated by aspiration of the substrate solution followed by washing for 10 minutes in distilled water at 25°C. The sections were counterstained with hematoxylin, dehydrated, and coverslipped, and images were digitized as with in situ hybridization. Specificity controls were performed by preabsorption of primary antibody with an excess of the antigen (1 g/mL in blocking solution, blocking peptides for IGFBP-1–6 were obtained from Santa Cruz Biotechnology Inc.). Preabsorption was performed overnight, and our standard immunochemistry protocol was then performed in parallel with slides being incubated either in primary antibody or primary antibody preabsorbed with excess antigen. Microscopy Ileal sections processed for either in situ hybridization or immunohistochemistry were immediately counterstained with hematoxylin, coverslipped, and imaged with an Olympus microscope using a 20× magnification objective. Digitized images were obtained using a Spot Jr. digital camera (Diagnostics, Inc.). Data Analysis Paired sections for each IGFBP (for both in situ hybridization and immunohistochemistry) were blinded by an independent party (J.B.P.), such that neither the histochemical technique nor the individual reagents were known to the grader (P.V.G.). The mucosal staining abundance for each section was then assessed with a modified binomial grading system as follows: mucosal staining equivalent to background = 0, mucosal staining above background = 0.5, abundant mucosal staining = 1.0, and highly abundant mucosal staining (evident by color saturation of the horseradish peroxidase substrate) = 2.0. The data were then unblinded, and the median and interquartile ranges were determined for each condition. The abundances were tabulated according to the following predefined definitions: 0 to 0.49 = background staining (−), 0.5 to 0.99 = mucosal staining greater than background (+/−), 1.0 to 1.99 = abundant mucosal staining (+), 2.0 = highly abundant mucosa staining (++). This nonlinear grading paradigm was felt to most accurately reflect the semiquantitative nature of comparative histochemistry. Significant differences between treatment conditions (defined as P ≤ 0.05) were determined by the Mann–Whitney U test. RESULTS Dexamethasone Increases Insulin-like Growth Factor Binding Protein-4 and -5 mRNA Abundance Daily intraperitoneal injections were comparably tolerated between dexamethasone-treated and untreated litters as previously reported (4). In situ hybridization of IGFBP transcripts revealed three distinct patterns of localization in control ileums at 3 days. IGFBP-l, -2, and -3 mRNA transcripts were widely distributed throughout the sections (Table 1 and Figs. 2A, C, E). IGFBP-4 and -6 mRNA transcripts were present above background and localized predominantly in the crypt epithelia (Table 1 and Figs. 2G, K). IGFBP-5 mRNA staining was faint and near background levels (Table 1 and Fig. 2I).TABLE 1: Mucosal abundance of insulin-like growth factor binding protein mRNA transcripts and peptides in the newborn mouse ileumFIG. 2.: Representative in situ hybridization of insulin-like growth factor binding proteins (IGFBPs) 1–6 in the ileum of control and dexamethasone-treated newborn mice. The arrangement is as follows: IGFBP-1: (A) control, (B) dexamethasone; IGFBP-2: (C) control, (D) dexamethasone; IGFBP-3: (E) control, (F) dexamethasone; IGFBP-4: (G) control, (H) dexamethasone; IGFBP-5: (I) control, (J) dexamethasone; and IGFBP-6: (K) control, (L) dexamethasone.Results from in situ hybridization performed in ileum from dexamethasone-treated mice were similar to those of controls in most cases. No differences in abundance were found between treatment conditions for IGFBP-1–3 and -6 in any of the animals (Table 1 and Figs. 2A–F, K–L). In contrast, there were modest categoric increases in both IGFBP-4 and -5 mRNA transcript abundance, although only IGFBP-4 was significantly different by the Mann–Whitney U test (Table 1 and Figs. 2G–J). Control hybridization, where riboprobe was omitted or the sections were preincubated with a cocktail of RNases, showed minimal to no detection (data not shown). Dexamethasone Increases the Abundance of Insulin-like Growth Factor Binding Proteins 2–5 in the Distal Villus Immunohistochemical localization of IGFBP-1, -2, -3, and -5 showed minimal staining in control ileum (Table 1 and Figs. 3A, D, G, M). In contrast, IGFBP-4 was faintly but reliably detected in epithelial cells along the distal portion of the villus in control ileum (Fig. 3J), and IGFBP-6 staining was abundant in all epithelial cells (Fig. 3P). Dexamethasone-treated ileum showed significant increases in the staining for IGFBP-2, -3, -4, and -5 within the distal villi when compared with controls (Table 1 and Fig. 3). This change was most evident for IGFBP-5 and was consistent across all animals. Because of the abrupt pattern change noted with these four IGFBPs, we did preabsorption controls in parallel to further confirm the specificity of the individual antibodies. Both preabsorption of primary antibody with competing antigen and omission of primary antibody demonstrates minimal background staining (Figs. 3F, I, L, O, R).FIG. 3.: Representative immunolocalization of insulin-like growth factor binding proteins (IGFBPs) 1–6 in the ileum of control and dexamethasone-treated newborn mice (with various methodology controls included in the third column). The arrangement is as follows: IGFBP-1: (A) control, (B) dexamethasone, (C) liver for positive control; IGFBP-2: (D) control, (E) dexamethasone, (F) IGFBP-2 preabsorbed dexamethasone; IGFBP-3: (G) control, (H) dexamethasone, (I) IGFBP-3 preabsorbed dexamethasone; IGFBP-4: (J) control, (K) dexamethasone, (L) IGFBP-4 preabsorbed dexamethasone; IGFBP-5: (M) control, (N) dexamethasone, (O) IGFBP-5 preabsorbed dexamethasone; IGFBP-6: (P) control, (Q) dexamethasone, (R) dexamethasone with primary antibody omitted to demonstrate background staining.IGFBP-l was not detected in either treatment condition (Table 1 and Figs. 3A, B), despite our demonstration of abundant mRNA with in situ hybridization (Table 1 and Figs. 2A, B). Immunolocalization of IGFBP-l in hepatocytes processed in parallel (Fig. 3C) suggests that the amount of IGFBP-l in the ileum is below the detection limit for the antiserum used. DISCUSSION In this study, we characterized the changing distribution and abundance of all six IGFBP mRNA transcripts and peptides within a model of dexamethasone-induced mucosal maturation. Our in situ hybridization data demonstrated that dexamethasone significantly increases IGFBP-4 mRNA transcript abundance. IGFBP-5 mRNA transcript abundance also showed a modest increase with dexamethasone treatment (we noted this to be particularly evident in the crypt epithelial cells), but this was not significantly different in our blinded assessment. In contrast, no change in abundance was evident with the other four IGFBP mRNA transcripts. Our immunolocalization data revealed significant increases in four IGFBPs (-2, -3, -4, and -5) after dexamethasone treatment. Of these, IGFBP-5 was the most dramatic (Table 1 and Fig. 3M, N). This finding is in contrast to the inhibitory effects of glucocorticoids on IGFBP-5 transcription in many in vitro models (including cultured osteoblasts, pituitary cells, and lung explants), leading us to suspect that IGFBP-5 is not directly governed by dexamethasone (14–16). However, IGFBP-5 may be obligatorily up-regulated during intestinal epithelial cell maturation, since progressive increases in IGFBP-5 expression and abundance have been demonstrated during lung epithelial cell development (17,18). While IGFBP-5 was found to have the greatest change in abundance and distribution, immunolocalization of IGFBP-2, -3, and -4 was also increased in the distal villi of dexamethasone-treated animals. Whether these four IGFBPs accumulate within epithelial cells through increased synthesis or uptake is not known, but their accumulation in dexamethasone-treated ileum parallels our previous observations of IGF-I redistribution from the mesenchyme to the mucosa (Fig. 1) (4,8). Taken together, our findings characterize the changing homeostasis of the IGF system during dexamethasone-induced maturation of the ileal mucosa and are consistent with at least three possible regulatory models: 1) accumulation of select IGFBPs within the mucosa draws IGF-I from the mesenchyme, 2) increasing IGF-I in the mucosa causes the accumulation of select IGFBPs (through synthesis, uptake and/or reduced proteolysis), or 3) the accumulation of IGF-I and one or more IGFBPs are coincident but are not causally related (e.g., IGFBP-5 transcription may be indirectly regulated by glucocorticoids, and IGFBP-5 peptide has been shown to have multiple effects that are independent of IGF-I (15,16)). These models are not exclusive and, given that four different IGFBPs were found to accumulate in the villus, each could prove to be true. There are some limitations and caveats to this study. First, although we did not find differences in mRNA transcript abundance for IGFBP-1, -2, -3, and -6, this semiquantitative technique is not sufficient to rule out small transcriptional changes between the two treatment conditions, particularly when an mRNA transcript is highly abundant in both conditions. Likewise, it is not clear that our increased immunolocalization of IGFBP-2, -3, -4, and -5 represents up-regulated translation. Increases in IGFBP peptide abundance could be secondary to changes in IGFBP-specific protease activity, since several reports have demonstrated that the abundances of individual IGFBPs are tightly governed by their respective proteases (1,17,18). Alternatively, dexamethasone-related increases in IGFBP immunolocalization could be unique to the suckling state. Theoretically, there could be enhanced lumenal uptake of these IGFBPs, since all four have been found in breast milk and could be accumulating because of induced maturation in digestive function (19–21). Finally, with regard to the IGF/growth axis in the postnatal ileum, the combined effect of these four IGFBPs within the mucosa cannot be assessed by this study. However, we have previously demonstrated that dexamethasone enhances mucosal growth in the newborn mouse ileum, and those findings occur in parallel with the IGFBP changes characterized in this study, suggesting a net increase in IGF-I availability. In summary, our findings are consistent with the hypothesis that dexamethasone redistributes IGF-I in the neonatal mouse ileum by changing the mucosal composition of IGFBPs. These findings further implicate the IGF system as a participant in mucosal maturation and, since precocious maturation is also seen in extremely premature infants who acquire ileal perforations, we suggest that IGFBPs may be mediators of the dexamethasone-related events that make these infants vulnerable to perforations. Acknowledgments: The authors thank Brian Brighton and Ward Jarvis for their technical assistance, Sarabeth Thomas, MS, for reading the manuscript and helpful discussions, and Jessica B. Paxton for the blinding of data.REFERENCES 1. Jones J, Clemmons DR. Insulin-like growth factors and their binding proteins: Biologic actions. Endo Rev 1995; 16:3–34. Cited Here 2. MacDonald RS. The role of insulin-like growth factors in small intestinal cell growth and development. Horm Metab Res 1999; 31:103–113. Cited Here | PubMed | CrossRef 3. Lund K. IGFs and the digestive tract. In: Rosenfield R, Roberts C, eds. Contemporary endocrinology: the IGF system. Totowa, NJ: Humana Press, 1998, pp 519–544. Cited Here 4. Gordon PV, Price WA, Stiles AD. Dexamethasone administration to newborn mice alters mucosal and muscular morphology in the ileum and modulates IGF-I localization. Pediatr Res 2001; 49:93–100. Cited Here | View Full Text | PubMed | CrossRef 5. Gordon PV, Rutledge J, Sawin R, Thomas S, Woodrurn D. Early postnatal dexamethasone increases the risk of focal small bowel perforation in extremely low birth weight infants. J Perinatal 1999; 19:573–577. Cited Here 6. Garland JS, Colleen AP, Pauly TH, et al. A three-day course of dexamethasone therapy to prevent chronic lung disease in ventilated neonates: A randomized trial. Pediatrics 1999; 104:91–99. Cited Here | View Full Text | PubMed | CrossRef 7. Stark AR, Carlo WA, Tyson JE, et al. Adverse effects of early dexamethasone treatment in extremely-low-birth-weight infants. N Engl J Med 2001; 344:95–101. Cited Here | View Full Text | PubMed | CrossRef 8. Gordon PV, Marshal DD, Stiles AD, Price WA. The clinical, morphologic, and molecular changes in the ileum associated with early postnatal dexamethasone administration: From the baby's bowel to the researcher's bench. Molec Genet Metab 2001; 72:91–103. Cited Here | PubMed | CrossRef 9. Retsch-Bogart GZ, Moats-Staats BM, Howard K, D'Ercole AJ, Stiles AD. Cellular localization of messenger RNAs for insulin-like growth factors (IGFs), their receptors and binding proteins during fetal rat lung development. Am J Respir Cell Mol Biol 1996; 14:61–69. Cited Here | PubMed | CrossRef 10. Moats-Staats BM, Price WA, Xu L, Jarvis W, Stiles AD. Regulation of the insulin-like growth factor system during normal rat lung development. Am J Respir Cell Mol Biol 1995; 12:56–64. Cited Here | PubMed | CrossRef 11. Busby WH, Snyder DK, Clemmons DR. Radioimmunoassay of a 26,000-dalton plasma insulin-like growthfactor-binding protein: Control by nutritional variables. J Clin Endocrinol Metab 1988; 67:1225–1230. Cited Here | PubMed 12. Sheikh MS, Shao ZM, Hussain A, et al. Regulation of insulin-like growth factor-binding-protein-1, 2,3,4,5, and 6: Synthesis, secretion, and gene expression in estrogen receptor-negative human breast carcinoma cells. J Cell Physiol 1993; 155:556–567. Cited Here | PubMed | CrossRef 13. Chevally T, Strond DD, Mohan S, Baylink D, Linkhart TA. Evidence for a role for insulin-like growth factor binding proteins in glucocorticoid inhibition of normal human osteoblast-like cell proliferation. Eur J Endocrinol 1996; 134:591–601. Cited Here | PubMed | CrossRef 14. Cheng SL, Zhang SF, Mohan S, et al. Regulation of insulin-like growth factors I and II and their binding proteins in human bone marrow stromal cells by dexamethasone. J Cell Biochem 1998; 71:449–458. Cited Here | PubMed | CrossRef 15. Fielder PJ, Tauber JP, Wilson KF, Pham HM, Rosenfeld RG. Insulin-like growth factors (IGFs) stimulate and dexamethasone inhibits IGF binding protein (EP)-5 expression in a mouse pituitary cell line. Growth Regul 1993; 3:226–234. Cited Here | PubMed 16. van de Wetering JK, Elfring RH, Oosterlaken-Dijksterhuis MA, Mol JA, Haagsman HP, Batenburg JJ. Perinatal expression of IGFEPs in rat lung and its hormonal regulation in fetal lung explants. Am J Physiol 1997; 273:Ll174–Ll181. Cited Here 17. Retsch-Bogart GZ, Moats-Staats EM, Howard K, D'Ercole AJ, Stiles AD. Cellular localization of messenger RNAs for insulin-like growth factors (IGFs), their receptors and binding proteins during fetal rat lung development. Am J Resp Cell Mol Biol 1996; 14:61–69. Cited Here | PubMed | CrossRef 18. Wallen LD, Myint W, Nygard K, Shimasake S, Clemmons DR, Han VK. Cellular distribution of insulin-like growth factor binding protein mRNAs and peptides during rat lung development. J Endocrinol 1997; 155:313–327. Cited Here | PubMed | CrossRef 19. Donovan SM, McNeil LK, Jimenez-Flores R, Odle J. Insulin-like growth factors and insu1in-like growth factor binding proteins in porcine serum and milk throughout lactation. Pediatr Res 1994; 36:159–168. Cited Here | View Full Text | PubMed | CrossRef 20. Gibson CA, Staley MD, Baumracker CR. Identification of IGF binding proteins in bovine milk and the demonstration of IGFEP-3 synthesis and release by bovine mammary epithelial cells. J Anim Sci 1999; 77:1547–1557. Cited Here | PubMed | CrossRef 21. Baumrucker CR, Erondu NE. Insulin-like growth factor (IGF) system in the bovine mammary gland and milk. J Mammary Gland Biol Neoplasia 2000; 5:53–64. Cited Here | View Full Text | PubMed Keywords:Dexamethasone; Insulin-like growth factor binding protein; Ileum; Bowel perforation © 2002 Lippincott Williams & Wilkins, Inc.View full article text


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