Early Disease Development Research Articles

Heterotypic Three-dimensional <em>In Vitro</em> Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades. Whilst stage I tumors have 5-year survival rates >85%, survival rates for stage III/IV disease are <40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF) cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Abstract 141: Bicuspid Aortic Valve Hemodynamic Abnormalities Promote Early Development of Calcific Aortic Valve Disease

INTRODUCTION: The bicuspid aortic valve (BAV) is the most common congenital cardiac anomaly and is frequently associated with calcific aortic valve disease (CAVD). Although CAVD also develops in the normal tricuspid aortic valve (TAV), its progression in the BAV is more rapid. While the accelerated calcification of BAV leaflets has been linked to genetic and atherogenic predispositions, hemodynamic abnormalities are increasingly pointed as potential pathogenic contributors. HYPOTHESIS: Supported by our previous work, which demonstrated the sensitivity of valve leaflets to the surrounding blood flow and associated wall-shear stress (WSS), we hypothesize that the abnormal WSS experienced by BAV leaflets contribute to CAVD development by promoting valvular inflammation, remodeling and osteogenic differentiation. OBJECTIVE: This study aims at comparing ex vivo the effects of TAV and BAV leaflet WSS on valvular pathogenesis. METHODS: The native, side-specific WSS experienced by TAV and type-I (i.e., fused and non-coronary) BAV leaflets were obtained computationally using fluid-structure interaction simulations. Fresh porcine leaflets were subjected for 48 hours to each of the three WSS conditions using a novel double-sided shear stress bioreactor. Tissue response was characterized via Western blot and immunohistochemistry in terms of markers of endothelial activation (VCAM-1, ICAM-1), paracrine expression (BMP-4), TGF-β/Wnt signaling pathways (TGF-β1, β-catenin), extracellular matrix remodeling (cathepsin L, MMP-2, MMP-9) and osteogenic differentiation (α-SMA, osteocalcin). RESULTS: No significant differences in VCAM-1 and ICAM-1 expressions were detected between tissue exposed to TAV and BAV WSS. While the native WSS experienced by the TAV and non-coronary BAV leaflets maintained tissue homeostasis, tissue exposure to the fused BAV leaflet WSS resulted in a significant pathological response marked by the upregulations of BMP-4, β-catenin, MMP-2 and osteocalcin expressions. CONCLUSION: This study demonstrates the pathological nature of the native BAV hemodynamics and confirms the higher susceptibility of the fused BAV leaflet to calcify. The results provide new insights into the hemodynamic theory of BAV calcification.

The Growing Epidemic of Hypertension Among Children and Adolescents: A Challenging Road Ahead

Currently, it is clear that primary hypertension begins in childhood and that it contributes to the early development of chronic kidney disease (CKD). Hypertension also increases the risk of cardiovascular morbidity and mortality, and that risk rises as blood pressure levels escalate. As among adult patients, overweight and obesity rates are on the rise among children and adolescents with primary hypertension and can develop target organ damage including left ventricular hypertrophy. An elevated level of C-reactive protein (CRP) and microalbuminuria are early manifestations of cardiovascular disease and CKD in hypertensive patients. Lifestyle interventions are recommended for all children with hypertension. Pharmacologic therapy should be added for symptomatic children, those with stage 2 hypertension, and children with prehypertension and stage 1 hypertension who exhibit an insufficient response to lifestyle modifications. Although the recommendations for choice of drugs generally are similar for children and adults, dosages for children should be lower, based on weight, and adjusted very carefully. Medications that are effective and safe for children and adolescents include thiazide diuretics, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, beta-blockers, and calcium channel-blockers. Hypertension is not being detected early enough for initiation of a treatment regimen to reduce death and disability. Initiatives should be undertaken to make health care providers and the general population more aware of the seriousness of hypertension in children and adolescents. This review focuses on the principles underlying the importance of a team approach for hypertension control, especially one that incorporates increased data sharing using enhanced health information technology for early detection and intervention.

Novel APP/Aβ mutation K16N produces highly toxic heteromeric Aβ oligomers

Here, we describe a novel missense mutation in the amyloid precursor protein (APP) causing a lysine-to-asparagine substitution at position 687 (APP770; herein, referred to as K16N according to amyloid-β (Aβ) numbering) resulting in an early onset dementia with an autosomal dominant inheritance pattern. The K16N mutation is located exactly at the α-secretase cleavage site and influences both APP and Aβ. First, due to the K16N mutation APP secretion is affected and a higher amount of Aβ peptides is being produced. Second, Aβ peptides carrying the K16N mutation are unique in that the peptide itself is not harmful to neuronal cells. Severe toxicity, however, is evident upon equimolar mixture of wt and mutant peptides, mimicking the heterozygous state of the subject. Furthermore, Aβ42 K16N inhibits fibril formation of Aβ42 wild-type. Even more, Aβ42 K16N peptides are protected against clearance activity by the major Aβ-degrading enzyme neprilysin. Thus the mutation characterized here harbours a combination of risk factors that synergistically may contribute to the development of early onset Alzheimer disease.

Trisomy for Synaptojanin1 in Down syndrome is functionally linked to the enlargement of early endosomes

Enlarged early endosomes have been observed in neurons and fibroblasts in Down syndrome (DS). These endosome abnormalities have been implicated in the early development of Alzheimer's disease (AD) pathology in these subjects. Here, we show the presence of enlarged endosomes in blood mononuclear cells and lymphoblastoid cell lines (LCLs) from individuals with DS using immunofluorescence and confocal microscopy. Genotype-phenotype correlations in LCLs carrying partial trisomies 21 revealed that triplication of a 2.56 Mb locus in 21q22.11 is associated with the endosomal abnormalities. This locus contains the gene encoding the phosphoinositide phosphatase synaptojanin 1 (SYNJ1), a key regulator of the signalling phospholipid phosphatidylinositol-4,5-biphosphate that has been shown to regulate clathrin-mediated endocytosis. We found that SYNJ1 transcripts are increased in LCLs from individuals with DS and that overexpression of SYNJ1 in a neuroblastoma cell line as well as in transgenic mice leads to enlarged endosomes. Moreover, the proportion of enlarged endosomes in fibroblasts from an individual with DS was reduced after silencing SYNJ1 expression with RNA interference. In LCLs carrying amyloid precursor protein (APP) microduplications causing autosomal dominant early-onset AD, enlarged endosomes were absent, suggesting that APP overexpression alone is not involved in the modification of early endosomes in this cell type. These findings provide new insights into the contribution of SYNJ1 overexpression to the endosomal changes observed in DS and suggest an attractive new target for rescuing endocytic dysfunction and lipid metabolism in DS and in AD.

Abstract P150: Aldosterone Dysregulation Increases Cardiovascular Risk in African-American Youth

Background: The prevalence of hypertension and blood pressure-related target organ damage in African-Americans is among the highest in the world. We hypothesize that this is in part due to aldosterone dysregulation among African-Americans beginning in youth and leading to the early development of cardiovascular disease in this population. To begin to test this hypothesis, we examined ethnic differences in aldosterone regulation in normal, healthy adolescents. Methods: The subjects in this study were 145 (60 Caucasian, 85 African-American) healthy, normotensive youth aged 15–19 years. Testing was performed following 72 hours on a controlled sodium diet. Testing consisted of the collection of aldosterone, systolic blood pressure (SBP), and urinary sodium excretion (U Na V) during continuous water intake (400 ml total) over a 2 hour period. An echocardiogram was also performed to measure target organ changes. Log transformations were performed on aldosterone levels prior to analyses. Results: African-American compared to Caucasian subjects had higher casual SBP (109±10 v 104±10; p=.006) and relative wall thickness (0.32±.03 v 0.34±.04; p=.003). During the testing procedure African-Americans also had lower levels of aldosterone (4.78±.6 v 4.35±.6 pg/ml; p =.001). In the Caucasian subjects only, aldosterone was inversely correlated with U Na V (r=−0.427; p=.001) and U Na V was positively correlated with SBP (r=0.356; p=.001). The subjects were divided into those in the upper and lower quartiles of salt intake for further analysis. The interaction between race and salt intake was significant for aldosterone (F=7.173; p=.008). Caucasian subjects with high salt intake had lower aldosterone (4.56±.59 v 5.02±0.59 pg/ml). However, aldosterone levels did not differ by salt intake in African-Americans. Summary and Conclusion: African-American subjects did not show the expected associations between aldosterone and the pressure natriuresis relation. Furthermore, African-Americans on the high salt intake failed to suppress aldosterone. These findings are consistent with our hypothesis that aldosterone dysregulation in youth may lead to the early development of cardiovascular disease in Africans-Americans.

Cytoprotective effects of hesperetin and hesperidin against amyloid β‐induced impairment of glucose transport through downregulation of neuronal autophagy

This study investigated whether flavonoids, such as hesperetin and hesperidin, inhibited amyloid β (Aβ)-impaired glucose utilization through regulating cellular autophagy in insulin-stimulated neuronal cells. In this study, we used a toxic Aβ1-42 peptide to impair insulin-stimulated glucose utilization in Neuro-2A cells, and this study also hypothesized that Aβ-induced autophagy might be emerging as a key process regulating neuronal glucose uptake. Additionally, hesperetin and hesperidin were used to test the neuroprotective effect against Aβ-induced impairment of glucose utilization. Our data found that Aβ-stimulated autophagy activation promoted the phenomenon of impairment of neuronal energy metabolism, including glucose uptake, glucose transporters (GLUTs), and insulin signaling cascades. In this study, confocal images of autophagy punctate further confirmed that downregulation of Aβ-stimulated autophagy could increase insulin-stimulated neuronal glucose uptake. Moreover, treatment with hesperetin and hesperidin improved Aβ-impaired glucose utilization by inhibiting Aβ-induced autophagy in neuronal cells. These findings suggest that downregulation of autophagy may be one of the approaches to control the impairment of energy metabolism leading to neuronal injury in the early development of Alzheimer's disease, and hesperetin or hesperidin may be a potential agent in the preventing of Alzheimer's disease progression.

The effect of maternal hypercholesterolemia on the placenta and fetal arteries in rabbits

To investigate the degree of placental permeability in dyslipidemic rabbits and the consequent vascular dysfunction in fetuses of female rabbits with high lipoprotein levels. Fifteen adult females New Zealand White rabbits were divided into two groups. Group 1(n=5) - hypercholesterolemic diet with 0.5% cholesterol, and Group 2 (n=10) - control. On day 30, the levels of plasma lipoproteins and triglycerides were analyzed in the mothers, and the presence of collagen was analyzed in the placenta as well as in fetal coronary and aorta. Statistical analyses used the Student's t and the Mann-Whitney tests. Lipoprotein levels were significantly different (p=0.02 to p<0.001) in experimental and control groups. In the hypercholesterolemic group, total cholesterol levels were in average 793 mg/dl; triglycerides were in average 257 mg/dl; HDL-C was 48 mg/dl, and LDL-C was in average 692 mg/dl. The amount of collagen per micrometers square (mµ²) in samples from hypercholesterolemic animals was significantly higher than in the control group. The study confirmed placental permeability to lipoproteins, shown by increased amounts of collagen in fetal tissues. This alteration results in increased susceptibility to atherosclerosis in adult life, representing a risk factor for the early development of disease, which may appear even in the prenatal period.

Human immunodeficiency virus and liver disease forum 2010: Conference proceedings

Liver disease continues to represent a critical mediator of morbidity and mortality in those with human immunodeficiency virus (HIV) infection. The frequent presence and overlap of concomitant injurious processes, including hepatitis C virus and hepatitis B virus infections, hepatoxicity associated with antiretroviral therapeutic agents, alcohol, and other toxins, in the setting of immunosuppression lead to rapid fibrotic progression and early development of end-stage liver disease. This conference summary describes the proceedings of a state-of-the-art gathering of international experts designed to highlight the status of current research in epidemiology, natural history, pathogenesis, and treatment of HIV and liver disease.

Blood Pressure and Vascular Alterations with Growth in Childhood

The long preclinical phase of atherosclerosis involves the interaction of genetic and environmental factors that modulate the progression of disease from early life. A variety of noninvasive tests have been used to study the arterial phenotype of childhood, allowing identification of arterial alterations long before clinical symptoms of cardiovascular (CV) disease become apparent. These techniques have improved our understanding of the evolution of atherosclerosis, indentifying three major developmental factors which influence the future risk of CV disease in childhood: prenatal growth, early postnatal growth and childhood obesity. Specific changes in arterial properties and increased values of blood pressure are detectable in children with low birth weight, suggesting that intrauterine growth retardation could programme the future risk of CV disease. However, an accelerated growth rate in infancy and early childhood is often associated with low birth weight and with specific vascular alterations, making it difficult to distinguish the contribution of prenatal and postnatal growth patterns to later cardiovascular disease risk. Relationships between growth patterns and CV disease risk are further complicated by the link between rapid postnatal growth and later development of childhood overweight and obesity, conditions associated with early changes in vascular physiology and that potentially affect future CV outcome. This article aims to provide an overview of evidence in support of fetal programming and growth acceleration hypotheses for adult CV disease. Specific attention is given to obesity-related vascular alterations and their effects on future CV disease risk. We also summarise current non-invasive approaches to investigate the precursors and early development of CV disease, emphasising their potential applications in childhood.

Immunotherapy of food allergy: what is effective?

Allergic asthma as a complex outcome of pathogenic immunological, cellular and functional modifications of the airways is initiated by allergic reactions as a response mostly to inhalant allergens. Dysregulation of innate and adaptive immune functions contribute to the pathogenesis of the disease. A hallmark is the development of a Th2-driven inflammatory response in the airways. The underlying Th2-skewed balance is the result of a multi-functional process in which genetic predisposition and environmental exposures interact as major players. Maturation of the immune system already starts in utero, the most critical phase in the ontogenetic programming of the offspring. Endogenous as well as exogenous exposures may influence the maturation and differentiation of immune cells of the fetus and may thereby contribute to disorders such as allergies and asthma later on in life. Epigenetic mechanisms are proposed to mediate these effects. A comprehensive overview on the interaction of fetal exposures and the developing immune system will be provided that may contribute to or protect the progeny against the development of asthma. The new and exciting field of epigenetics will be highlighted with respect to T-cell differentiation and early allergic disease development. Furthermore, we emphasize new investigations that aimed to analyze fetal host innate immune responses to environmental microbial microorganisms and their possible future application in asthma protection.

A follow-up study of peri-implantitis cases after treatment

The aim of this retrospective study was to follow patient cases in a longitudinal manner after peri-implantitis treatment. Two hundred and eighty-one patient cases were selected consecutively from the archives of the Oral Microbiological Diagnostic Laboratory, Gothenburg, Sweden based on microbial analysis of bacterial samples taken from diseased implants. It was feasible to follow-up 245 patients after treatment for a period ranging from 9 months to 13 years. In 54.7% of the patients it was not feasible to arrest progression of peri-implantitis. Smoking and smoking dose were found to be significantly correlated to failure of peri-implantitis treatment (p<0.05). Early disease development was also significantly associated with failure (p<0.05). Bone plasty in conjunction to antibiotics during surgery was significantly associated with arrested lesions (p<0.05). In a multiple regression model disease development was the only independent variable to significantly predict the likelihood of treatment success. Peri-implant health may not be easy to establish, especially in cases that develop disease early. Homogenous treatment protocols rather than empirical treatment attempts should be adopted.

Jejunostomy after oesophagectomy: A review of evidence and current practice

Patients undergoing oesophagectomy often have nutritional needs at the time of diagnosis and in the post-operative period. The aim of this article is to review the current literature and report on the author's experience of routine feeding jejunostomy insertion following oesophagectomy. The records of forty-eight consecutive patients undergoing oesphagectomy under the author's care were reviewed. Although the evidence of benefit of peri-operative feeding in patients undergoing oesophagectomy is limited, there is a clear need to establish a feeding route at the time of surgery. Oesophagectomy is associated with a mortality rate of 5-10% and a morbidity rate of 30-40% even in high-volume specialist centres. Over 50% of patients developing complications will require an alternative to oral feeding beyond 30 d. The enteral route is preferred in terms of safety and cost. A surgical feeding jejunostomy is associated with a low complication rate and a mortality rate of less than 1%. In forty-eight patients undergoing oesophagectomy the average weight loss at 6 months was 8·4 kg with only 8% regaining their pre-operative weight. Large reductions in weight at 6 months post-operatively were recorded irrespective of the development of post-operative complications or early recurrent disease. Routine jejunostomy insertion is recommended to ensure adequate nutrition in patients who develop post-operative complications and for those patients with long-term reduced appetite and poor oral intake.

Adiposity, fitness and metabolic risk in children: A cross-sectional and longitudinal study

To better understand the early development of cardiovascular disease, we examined the association of adiposity measures and cardiorespiratory fitness (CRF) with baseline values and changes in low-grade chronic inflammation and insulin resistance (IR) in school children. Eighty-three 1(st) and 5(th) grade children from a randomly selected control group of a physical activity intervention study (KISS) were prospectively studied during one academic year. Outcome variables included highly sensitive C-reactive protein (hs-CRP) and homeostasis assessment of IR (HOMA-IR). Adiposity measures included body mass index (BMI), waist circumference and the sum of skinfold thickness at four sites. CRF was assessed by the 20 m shuttle run test. All models were adjusted for age group, sex and pubertal group and included children who had a complete valid dataset for all variables. Baseline metabolic markers correlated positively with measures of adiposity and inversely with CRF (all p ≤ 0.02). On average, longitudinal changes in hs-CRP concentration increased with high baseline BMI in boys and skinfold thickness in both genders (both p < 0.01). Increases in adiposity had an additional effect on these changes (adjusted R(2) of the models including baseline and change in adiposity 0.49-0.53, p < 0.001). On the other hand, longitudinal changes in HOMA-IR were positively related to changes in BMI and waist circumference (p ≤ 0.03). Increases in HOMA-IR, but not in hs-CRP concentrations, were associated with low baseline CRF (p = 0.01). The relationship of baseline adiposity and CRF with increases in metabolic risk markers may have relevant implications for long-term cardiovascular risk. International Standard Randomised Controlled Trial Number (ISRCTN): isrctn.org 15360785.

Fatal Coronary Artery Disease in an Infant With Severe Mucopolysaccharidosis Type I

A previously healthy 10-month-old boy was referred to our hospital because of coarse facial features that were suggestive of lysosomal storage disease. Apart from noisy respiration, there was no medical history. Elevated levels of urinary glycosaminoglycans and complete deficiency of leukocyte α-l-iduronidase indicated severe mucopolysaccharidosis type I. A chest radiograph revealed a markedly enlarged heart, and echocardiography revealed hypertrophic cardiomyopathy. While hematopoietic stem cell transplantation was being planned, progressive cardiac failure developed with a striking hypokinesia of the left-ventricle free wall. In combination with ischemic changes on the electrocardiogram, this was suggestive of coronary artery disease. Results of coronary echo Doppler interrogation were inconclusive, and intravascular ultrasound in this little infant was not feasible. Despite the patient's small size, a successful selective coronary angiography was performed and revealed diffuse narrowing of the left coronary artery with collateral flow from the right coronary artery. Enzyme-replacement therapy was started immediately in an attempt to improve myocardial performance. Evaluation after 3 months, however, revealed complete obliteration of the left coronary main stem with diffuse hypokinesia/akinesia of the left ventricle. At the age of 13 months the boy died of terminal cardiac failure. This case report illustrates the importance of considering early development of coronary artery disease in children with severe mucopolysaccharidosis type I and cardiomyopathy.

Blood Lipid Levels and Injury to DNA in Obese Children

Obesity condition implies a significant health risk, and its association to other factors as dislipidemia and metabolic syndrome is the basis of several chronic non-communicable diseases. All of these elements have been lately associated to the impairment of DNA structure, which represents a critical menace to cellular function. The study of damage to DNA in obese children should be the basis for the administration of physical activity in early stages of life, due to its role as a major antioxidant. PURPOSE: To determine DNA damage and blood lipid levels in sedentary obese children. METHODS: Nine sedentary obese boys, 9.5 ± 1.3 years of age, 53.4 ± 8.4 kg of body weight, 140.3 ± 5.9 cm of height and 27.8 ± 3.4 kg/m2 of body mass index (BMI) were included. Not clinical evidence of other disease was detected. Oxidative damage to DNA was determined by the number of apurinic-apyrimidinic sites (APS) × 105. Plasma lipid levels by colorimetric-enzymatic procedure were measured after a 12 hour fast. Lipids were correlated to APS and were also compared with other reported values in some studies with obese children. RESULTS: Children's BMI was higher than 97 percentile of CDC charts and classified as severe obesity according to National Institute of Health in Mexico (INSP, 2006). The number of detected APS was 4.6 ± 4.4 × 105. Blood lipid levels were as follows: 189.3 ± 29.6 mg·dl-1 of total cholesterol (TC), 82.4 ± 35.7 mg·dl-1 of triglycerides (TG), 117.3 ± 23 mg·dl-1 of LDL-cholesterol (LDL) and 50.4 ± 11.5 mg·dl-1 of HDL-cholesterol (HDL). Values of TC, TG, LDL and HDL were adjusted by Z score according to those described in studies with obese children in Great Britain, Mexico and USA. TC, TG, and LDL were higher, while HDL was lower than those reported for children in the same range of age. No correlation between APS and lipids was observed. CONCLUSIONS: Oxidative damage in DNA, evidenced by the number of APS, and high blood lipid levels found in sedentary obese children may suppose an increased possibility for early development of chronic non-communicable diseases. More studies must be done to understand any association between these factors and should be the basis to perform physical activity programs which can change such risk, due to its antioxidant action.

Abstract 3007: Integrative analysis of histologically distinct oral lesions obtained from a single disease field uncovers stage-specific molecular alterations

Abstract Oral cancer (OC) is a leading cause of cancer death worldwide, with late stage diagnosis and high recurrence rates accounting for poor survival statistics. Like many solid tumors, OC develops through a series of histopathological stages: from hyperplasia to dysplasia to carcinoma in situ (CIS) and finally to invasive cancer. While dysplastic lesions can be readily detected in clinic, they are rarely treated as only a small fraction will ultimately progress to invasive disease. By identifying the molecular mechanisms driving early malignant disease, novel tools can be developed for discriminating progression risk in early lesions and identify druggable targets that could greatly improve currently stagnant oral cancer survival rates. Objective: To identify genes critical to early oral tumorigenesis that may also have utility as biomarkers for disease outcomes. Methods: Fluorescence visualization techniques were used to define OC disease fields. For each patient multiple biopsies revealing distinct disease stages were simultaneously collected from within the disease field. DNA copy number (SMRT CGH), methylation (Illumina Infinium), and gene expression (Agilent 4×44K) profiles on all collected premalignant biopsies from each patient (hyperplasia, dysplasia, and CIS) were generated. Gene candidates were identified from integrated datasets based on current dogma. For example, genes exhibiting decreased expression and concurrent increased methylation, and vice versa, were flagged for further analysis. Recurring genetic and signaling cascade events were defined using Ingenuity Pathway Analysis software and validated by sequencing and expression analysis. Results: While no DNA copy number changes were observed in dysplastic biopsies, several changes were observed in DNA methylation and gene expression data. This suggests that methylation changes may play a critical role in gene deregulation for early oral lesions. As expected, methylation and expression alterations were observed to increase with disease severity. Several genes were found to be deregulated at a frequency of &amp;gt;50% in dysplastic lesions, strongly suggesting a critical role for these candidates in early disease development. Additional genes were recurrently altered during progression to CIS. Pathway analysis of recurrent genes candidates revealed a potential role of the wnt/β-catenin pathway and inflammation/immune response pathways in oral lesion development. Conclusions: By leveraging high throughput platforms and a rare collection of patient-matched oral precancer tissues, we have shown that aberrant methylation is a critical mechanism driving molecular dysregulation in early oral tumorigenesis. Specific candidates identified by our integrative approach may have utility as biomarkers or therapeutic targets, ultimately helping to improve oral cancer outcomes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3007. doi:10.1158/1538-7445.AM2011-3007

Clinical diagnosis of metabolic and cardiovascular risks in overweight children: early development of chronic diseases in the obese child

Childhood overweight (body mass index (BMI)>90th centile) poses a major public health problem in so far as adult diseases manifest themselves already during childhood. In this review, after examining the prevalence of metabolic and cardiovascular diseases, the issue of whether BMI or other clinical parameters are valid tools to predict co-morbidities in children is discussed. Data of 2001-2008 are reviewed, including several studies conducted on more than 260,000 overweight and obese children in Germany and Switzerland. Apart from non-metabolic co-morbidities, namely musculoskeletal complications and attention deficit/hyperactivity disorders in up to 74% and up to 58% of overweight children, respectively, at least one cardiovascular and metabolic risk factor was seen in 52% of the overweight children, mostly high blood pressure (35%) with increased left ventricular mass or arterial stiffness. Signs of fatty liver disease or dyslipidemia were found in up to 29 and 32% of the children, respectively. Type 2 diabetes was found in less than 0.7% of the children, and an elevated fasting glucose or glucose intolerance in 3%. Irrespective of BMI, the quality of fat and protein intake predicted hypertension or insulin resistance, and fructose predicted lower (and more atherogenic) particle size of low-density lipoprotein-cholesterol. Out of the adiposity markers, waist circumference was closely correlated with insulin resistance and the components of the metabolic syndrome. In conclusion, risk factors are found in more than half of the overweight children, most frequently high blood pressure or dyslipidemia, and were mainly related to waist circumference, but also to BMI and fat mass. Even in the presence of normal BMI, screening for cardiovascular risk factors is advocated in each child with elevated waist circumference, but its cut-off points still remain to be validated. The increased occurrence of orthopedic and psychiatric complaints may detrimentally influence health-related lifestyle and obesity therapy.

Efficient Differentiation of Human Embryonic Stem Cells into Functional Cerebellar-Like Cells

The cerebellum has critical roles in motor and sensory learning and motor coordination. Many cerebellum-related disorders indicate cell therapy as a possible treatment of neural loss. Here we show that application of inductive signals involved in early patterning of the cerebellar region followed by application of different factors directs human embryonic stem cell differentiation into cerebellar-like cells such as granule neurons, Purkinje cells, interneuron, and glial cells. Neurons derived using our protocol showed a T-shaped polarity phenotype and express similar markers to the developed human cerebellum. Electrophysiological measurements confirmed functional electrical properties compatible with these cells. In vivo implantation of differentiated human embryonic stem cells transfected with MATH1-GFP construct into neonatal mice resulted in cell migration across the molecular and the Purkinje cell layers and settlement in the internal molecular layers. Our findings demonstrate that the universal mechanisms involved in the development of cerebellum can be efficiently recapitulated in vitro, which enables the design of new strategies for cell replacement therapy, to study early human development and pathogenesis of neurodegenerative diseases.

Asymptomatic 5-year-old child with hypothyroidism and concurrent severe hypercholesterolemia

Homozygous familial hypercholesterolemia (FH) affects 1 in 1 million children, whereas heterozygous FH affects 1 in 500 children. The concentration of low-density lipoprotein cholesterol (LDL-C) usually exceeds 500 mg/dL in affected children. If left untreated, these children are at risk for the development of atherosclerosis and early cardiovascular disease. However, very high LDL-C is not always the result of FH, and secondary causes of hypercholesterolemia should be ruled out as part of the medical workup, even though these causes are unusual in children.