A novel mutation in the glycogen synthase 2 gene in a child with glycogen storage disease type 0

Background: Glycogen storage disease type 0 is an autosomal recessive disease presenting in infancy or early childhood and characterized by ketotic hypoglycemia after prolonged fasting and postprandial hyperglycemia and hyperlactatemia. Sixteen different mutations have been identified to date in the gene which encodes hepatic glycogen synthase, resulting in reduction of glycogen storage in the liver. Case Presentation: Biochemical evaluation as well as direct sequencing of exons and exon-intron boundary regions of the GYS2 gene were performed in a patient presenting fasting hypoglycemia and postprandial hyperglycemia and her parents. The patient was found to be compound heterozygous for one previously reported nonsense mutation (c. 736 C>T; R243X) and a novel frameshift mutation (966_967delGA/insC) which introduces a stop codon 21 aminoacids downstream from the site of the mutation that presumably leads to loss of 51% of the COOH-terminal part of the protein. The glycemia and lactatemia of the parents after an oral glucose tolerance test were evaluated to investigate a possible impact of the carrier status on the metabolic profile. The mother, who presented a positive family history of type 2 diabetes, was classified as glucose intolerant and the father, who did not exhibit metabolic changes after the glucose overload, had an antecedent history of hypoglycemia after moderate alcohol ingestion. Conclusion: The current results expand the spectrum of known mutations in GYS2 and suggest that haploinsufficiency could explain metabolic abnormalities in heterozygous carriers in presence of predisposing conditions.

Estimating cardiovascular risk in patients with type 2 diabetes: a national multicenter study in Brazil

According to Brazilian National Data Survey diabetes is the fifth cause for hospitalization and is one of the ten major causes of mortality in this country. Aims to stratify the estimated cardiovascular risk (eCVR) in a population of type 2 diabetics (T2DM) according to the Framingham prediction equations as well as to determine the association between eCVR with metabolic and clinical control of the disease. Methods From 2000 to 2001 a cross-sectional multicenter study was conducted in 13 public out-patients diabetes/endocrinology clinics from 8 Brazilian cities. The 10-year risk of developing coronary heart disease (CHD) was estimated by the prediction equations described by Wilson et al (Circulation 1998). LDL equations were preferably used; when patients missed LDL data we used total cholesterol equations instead. Results Data from 1382 patients (59.0% female) were analyzed. Median and inter-quartile range (IQ) of age and duration of diabetes were 57.4 (51-65) and 8.8 (3-13) years, respectively without differences according to the gender. Forty-two percent of these patients were overweight and 35.4% were obese (the prevalence of higher BMI and obesity in this T2DM group was significantly higher in women than in men; p < 0.001). The overall estimated eCVR in T2DM patients was 21.4 (13.5-31.3). The eCVR was high (> 20%) in 738 (53.4%), intermediate in 202 (14.6%) and low in 442 (32%) patients. Men [25.1(15.4-37.3)] showed a higher eCVR than women [18.8 (12.4-27.9) p < 0.001]. The most common risk factor was high LDL-cholesterol (80.8%), most frequently found in women than in men (p = 0.01). The median of risk factors present was three (2-4) without gender differences. Overall we observed that 60 (4.3%) of our patients had none, 154(11.1%) one, 310 (22.4%) two, 385 (27.9%) three, 300 (21.7%) four, 149 (10.5%) five and six, (2%) six risk factors. A higher eCVR was noted in overweight or obese patients (p = 0.01 for both groups). No association was found between eCVR with age or a specific type of diabetes treatment. A correlation was found between eCVR and duration of diabetes (p < 0.001), BMI (p < 0.001), creatinine (p < 0.001) and triglycerides levels (p < 0.001) but it was not found with HbA1c, fasting blood glucose and postprandial glucose. A higher eCVR was observed in patients with retinopathy (p < 0.001) and a tendency in patients with microalbuminuria (p = 0.06). Conclusion: our study showed that in this group of Brazilian T2DM the eCVR was correlated with the lipid profile and it was higher in patients with microvascular chronic complications. No correlation was found with glycemic control parameters. These data could explain the failure of intensive glycemic control programs aiming to reduce cardiovascular events observed in some studies.

Characterization of Dengue Virus Type 2: New Insights on the 2010 Brazilian Epidemic

Dengue viruses (DENV) serotypes 1, 2, and 3 have been causing yearly outbreaks in Brazil. In this study, we report the reintroduction of DENV2 in the coast of Sao Paulo State. Partial envelope viral genes were sequenced from eighteen patients with dengue fever during the 2010 epidemic. Phylogenetic analysis showed this strain belongs to the American/Asian genotype and was closely related to the virus that circulated in Rio de Janeiro in 2007 and 2008. The phylogeny also showed no clustering by clinical presentation, suggesting that the disease severity could not be explained by distinct variants or genotypes. The time of the most recent common ancestor of American/Asian genotype and the Sao Paulo and Rio de Janeiro (SP/RJ) monophyletic cluster was estimated to be around 40 and 10 years, respectively. Since this virus was first identified in Brazil in 2007, we suggest that it was already circulating in the country before causing the first documented outbreak. This is the first description of the 2010 outbreak in the State of Sao Paulo, Brazil, and should contribute to efforts to control and monitor the spread of DENVs in endemic areas.

TCF7L2 variant genotypes and type 2 diabetes risk in Brazil: significant association, but not a significant tool for risk stratification in the general population

Background: Genetic polymorphisms of the TCF7L2 gene are strongly associated with large increments in type 2 diabetes risk in different populations worldwide. In this study, we aimed to confirm the effect of the TCF7L2 polymorphism rs7903146 on diabetes risk in a Brazilian population and to assess the use of this genetic marker in improving diabetes risk prediction in the general population. Methods: We genotyped the single nucleotide polymorphisms (SNP) rs7903146 of the TCF7L2 gene in 560 patients with known coronary disease enrolled in the MASS II (Medicine, Angioplasty, or Surgery Study) Trial and in 1,449 residents of Vitoria, in Southeast Brazil. The associations of this gene variant to diabetes risk and metabolic characteristics in these two different populations were analyzed. To access the potential benefit of using this marker for diabetes risk prediction in the general population we analyzed the impact of this genetic variant on a validated diabetes risk prediction tool based on clinical characteristics developed for the Brazilian general population. Results: SNP rs7903146 of the TCF7L2 gene was significantly associated with type 2 diabetes in the MASS-II population (OR = 1.57 per T allele, p = 0.0032), confirming, in the Brazilian population, previous reports of the literature. Addition of this polymorphism to an established clinical risk prediction score did not increased model accuracy (both area under ROC curve equal to 0.776). Conclusion: TCF7L2 rs7903146 T allele is associated with a 1.57 increased risk for type 2 diabetes in a Brazilian cohort of patients with known coronary heart disease. However, the inclusion of this polymorphism in a risk prediction tool developed for the general population resulted in no improvement of performance. This is the first study, to our knowledge, that has confirmed this recent association in a South American population and adds to the great consistency of this finding in studies around the world. Finally, confirming the biological association of a genetic marker does not guarantee improvement on already established screening tools based solely on demographic variables.

Hypotheses, rationale, design, and methods for prognostic evaluation in type 2 diabetic patients with angiographically normal coronary arteries. The MASS IV-DM Trial

Background: The MASS IV-DM Trial is a large project from a single institution, the Heart Institute (InCor), University of Sao Paulo Medical School, Brazil to study ventricular function and coronary arteries in patients with type 2 diabetes mellitus. Methods/Design: The study will enroll 600 patients with type 2 diabetes who have angiographically normal ventricular function and coronary arteries. The goal of the MASS IV-DM Trial is to achieve a long-term evaluation of the development of coronary atherosclerosis by using angiograms and coronary-artery calcium scan by electron-beam computed tomography at baseline and after 5 years of follow-up. In addition, the incidence of major cardiovascular events, the dysfunction of various organs involved in this disease, particularly microalbuminuria and renal function, will be analyzed through clinical evaluation. In addition, an effort will be made to investigate in depth the presence of major cardiovascular risk factors, especially the biochemical profile, metabolic syndrome inflammatory activity, oxidative stress, endothelial function, prothrombotic factors, and profibrinolytic and platelet activity. An evaluation will be made of the polymorphism as a determinant of disease and its possible role in the genesis of micro- and macrovascular damage. Discussion: The MASS IV-DM trial is designed to include diabetic patients with clinically suspected myocardial ischemia in whom conventional angiography shows angiographically normal coronary arteries. The result of extensive investigation including angiographic follow-up by several methods, vascular reactivity, pro-thrombotic mechanisms, genetic and biochemical studies may facilitate the understanding of so-called micro- and macrovascular disease of DM.

Expression of matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in medullary thyroid carcinoma: Prognostic implications

Background: Metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) participate in the degeneration of the extracellular matrix and are associated with carcinogenesis. MMP-2 is one of the main metalloproteinases active in neoplasia and is a marker of the malignant phenotype. Since the biological behavior of medullary thyroid carcinoma (MTC) varies widely, the present study was undertaken to determine if there is a correlation between the clinical evolution of MTC and the immunohistochemically detected expression of these enzymes in thyroid surgical specimens containing MTC. If so, their expression would be a novel indicator of the prognosis of MTC. Methods: Thirty-seven patients with MTC who had undergone thyroid surgery were followed for an average of 73 months. Immunohistochemical staining for metalloproteinase-related enzymes was performed in surgical paraffin blocks. The clinical status of the patients after surgery and at the end of the study period was characterized to determine correlations between these and the immunohistochemical markers. A value of p < 0.05 was considered statistically significant. Results: At the end of the study period, 15 patients (40.5%) were alive and without evidence of MTC, 17 (45.9%) had persistent MTC, and 5 (13.5%) had a relapse of their neoplasia. Four patients (10.8%) died during the course of the study. There was a significant correlation (p = 0.0005) between the immunohistochemical staining for MMP-2 and the clinical condition of the patients at the end of the study period, and a correlation between the state of apparent cure compared to persistence of MTC after thyroid surgery (p = 0.0207). No significant correlations were observed between either TIMP-2 expression or immune marking of metastatic lymph nodes and the clinical variables studied. Conclusion: Immunohistochemical expression of MMP-2 in thyroid surgical specimens from patients with MTC is a novel indicator of the prognosis of this cancer.

The International Collaboration on Air Pollution and Pregnancy Outcomes: Initial Results

BACKGROUND: The findings of prior studies of air pollution effects on adverse birth outcomes are difficult to synthesize because of differences in study design. OBJECTIVES: The International Collaboration on Air Pollution and Pregnancy Outcomes was formed to understand how differences in research methods contribute to variations in findings. We initiated a feasibility study to a) assess the ability of geographically diverse research groups to analyze their data sets using a common protocol and b) perform location-specific analyses of air pollution effects on birth weight using a standardized statistical approach. METHODS: Fourteen research groups from nine countries participated. We developed a protocol to estimate odds ratios (ORs) for the association between particulate matter <= 10 mu m in aerodynamic diameter (PM(10)) and low birth weight (LBW) among term births, adjusted first for socioeconomic status (SES) and second for additional location-specific variables. RESULTS: Among locations with data for the PM(10) analysis, ORs estimating the relative risk of term LBW associated with a 10-mu g/m(3) increase in average PM(10) concentration during pregnancy, adjusted for SES, ranged from 0.63 [95% confidence interval (CI), 0.30-1.35] for the Netherlands to 1.15 (95% CI, 0.61-2.18) for Vancouver, with six research groups reporting statistically significant adverse associations. We found evidence of statistically significant heterogeneity in estimated effects among locations. CONCLUSIONS: Variability in PM(10)-LBW relationships among study locations remained despite use of a common statistical approach. A more detailed meta-analysis and use of more complex protocols for future analysis may uncover reasons for heterogeneity across locations. However, our findings confirm the potential for a diverse group of researchers to analyze their data in a standardized way to improve understanding of air pollution effects on birth outcomes.

Prognostic index expression of cyclin-D1, cerbB-2 and VEGF: metastases vs corresponding primary cancers and metastatic vs non-metastatic adenocarcinomas

The prognostic relevance of different molecular markers in lung cancer is a crucial issue still worth investigating, and the specimens collected and analyzed represent a valuable source of material. Cyclin-D1, c-erbB-2 and vascular endothelial growth factor (VEGF) have shown to be promising as prognosticators in human cancer. In this study, we sought to examine the importance of Cyclin-D1, c-erbB-2 and VEGF, and to study the quantitative relationship among these factors and disease progression in metastases vs corresponding primary cancer, and metastatic vs non metastatic cancers. Material and Methods: We used immunohistochemistry and morphometric analysis to evaluate the amount of tumour staining for Cyclin-D1, c-erbB-2 and VEGF in 52 patients with surgically excised ademocarcinoma of the lung, and the outcome for our study was survival time until death from hematogenic metastases. Results: Metastasis presented lower c-erbB-2 expression than corresponding primary cancers (p=0.02). Cyclin-D1 and VEGF expression were also lower in metastases than in corresponding primary cancers, but this difference did not achieve statistical significance. Non-metastatic cancers also presented significantly lower Cyclin-D1 and c-erbB-2 expression than metastatic cancers (p<0.01 and p<0.01, respectively). Equally significant was the difference between higher c-erbB-2 expression by metastatic cancers compared to non-metastatic cancers (p=0.02). Considering survival in Kaplan-Maier analysis, Cyclin-D1 (p=0.04), c-erbB-2 (p=0.04) and VEGF (p<0.01) were important predictors of survival in metastatic cancers.

Opportunity for catch-up HPV vaccination in young women after first delivery

Background Early age at first delivery has been identified as a risk factor for high-risk HPV-type infection and cervical cancer development. Methods A cross-sectional study was carried out in a large public maternity hospital in Sao Paulo, Brazil. During June 2006 to February 2007, 301 women aged 15-24 years who gave birth to their first child were recruited between 43 and 60 days after delivery. Detection of HPV DNA in cervical specimens was performed using a standardised PCR protocol with PGMY09/11 primers. The association of selected factors with HPV infection was assessed by using a Generalised Linear Model. Results HPV DNA was detected in 58.5% (95% CI 52.7% to 64.0%) of the enrolled young women. The most common types of HPV found were: HPV16, HPV51, HPV52, HPV58 and HPV71. The overall prevalence of HPV types targeted by the HPV prophylactic vaccines was: HPV 16-12.0%, HPV 18-2.3% and HPV 6 and 11 4.3%. In the multivariate analysis, only age (inversely, p for trend=0.02) and smoking habits were independently associated with HPV infection. Conclusions The findings show that these young primiparous women had high cervical HPV prevalence, suggesting that this is a high-risk group for cervical cancer development. Nevertheless, 17.3% were positive for any of the four HPV types included in HPV vaccines (HPV6, 11, 16 or 18), with 13.3% positive for HPV 16 or 18 and only 1.0% having both vaccine related-oncogenic HPV types. Thus, young primiparous women could benefit from catch-up HPV vaccination programmes.

Sensitization Prevalence, Antibody Cross-Reactivity and Immunogenic Peptide Profile of Api g 2, the Non-Specific Lipid Transfer Protein 1 of Celery

Background: Celery (Apium graveolens) represents a relevant allergen source that can elicit severe reactions in the adult population. To investigate the sensitization prevalence and cross-reactivity of Api g 2 from celery stalks in a Mediterranean population and in a mouse model. Methodology: 786 non-randomized subjects from Italy were screened for IgE reactivity to rApi g 2, rArt v 3 (mugwort pollen LTP) and nPru p 3 (peach LTP) using an allergen microarray. Clinical data of 32 selected patients with reactivity to LTP under investigation were evaluated. Specific IgE titers and cross-inhibitions were performed in ELISA and allergen microarray. Balb/c mice were immunized with purified LTPs; IgG titers were determined in ELISA and mediator release was examined using RBL-2H3 cells. Simulated endolysosomal digestion was performed using microsomes obtained from human DCs. Results: IgE testing showed a sensitization prevalence of 25.6% to Api g 2, 18.6% to Art v 3, and 28.6% to Pru p 3 and frequent co-sensitization and correlating IgE-reactivity was observed. 10/32 patients suffering from LTP-related allergy reported symptoms upon consumption of celery stalks which mainly presented as OAS. Considerable IgE cross-reactivity was observed between Api g 2, Art v 3, and Pru p 3 with varying inhibition degrees of individual patients' sera. Simulating LTP mono-sensitization in a mouse model showed development of more congruent antibody specificities between Api g 2 and Art v 3. Notably, biologically relevant murine IgE cross-reactivity was restricted to the latter and diverse from Pru p 3 epitopes. Endolysosomal processing of LTP showed generation of similar clusters, which presumably represent T-cell peptides. Conclusions: Api g 2 represents a relevant celery stalk allergen in the LTP-sensitized population. The molecule displays common B cell epitopes and endolysosomal peptides that encompass T cell epitopes with pollen and plant-food derived LTP.

Expression of Monocarboxylate Transporters 1, 2, and 4 in Human Tumours and Their Association with CD147 and CD44

Monocarboxylate transporters (MCTs) are important cellular pH regulators in cancer cells; however, the value of MCT expression in cancer is still poorly understood. In the present study, we analysed MCT1, MCT2, and MCT4 protein expression in breast, colon, lung, and ovary neoplasms, as well as CD147 and CD44. MCT expression frequency was high and heterogeneous among the different tumours. Comparing with normal tissues, there was an increase in MCT1 and MCT4 expressions in breast carcinoma and a decrease in MCT4 plasma membrane expression in lung cancer. There were associations between CD147 and MCT1 expressions in ovarian cancer as well as between CD147 and MCT4 in both breast and lung cancers. CD44 was only associated with MCT1 plasma membrane expression in lung cancer. An important number of MCT1 positive cases are negative for both chaperones, suggesting that MCT plasma membrane expression in tumours may depend on a yet nonidentified regulatory protein.

Novel Primate-Specific Genes, RMEL 1, 2 and 3, with Highly Restricted Expression in Melanoma, Assessed by New Data Mining Tool

Melanoma is a highly aggressive and therapy resistant tumor for which the identification of specific markers and therapeutic targets is highly desirable. We describe here the development and use of a bioinformatic pipeline tool, made publicly available under the name of EST2TSE, for the in silico detection of candidate genes with tissue-specific expression. Using this tool we mined the human EST (Expressed Sequence Tag) database for sequences derived exclusively from melanoma. We found 29 UniGene clusters of multiple ESTs with the potential to predict novel genes with melanoma-specific expression. Using a diverse panel of human tissues and cell lines, we validated the expression of a subset of three previously uncharacterized genes (clusters Hs.295012, Hs.518391, and Hs.559350) to be highly restricted to melanoma/melanocytes and named them RMEL1, 2 and 3, respectively. Expression analysis in nevi, primary melanomas, and metastatic melanomas revealed RMEL1 as a novel melanocytic lineage-specific gene up-regulated during melanoma development. RMEL2 expression was restricted to melanoma tissues and glioblastoma. RMEL3 showed strong up-regulation in nevi and was lost in metastatic tumors. Interestingly, we found correlations of RMEL2 and RMEL3 expression with improved patient outcome, suggesting tumor and/or metastasis suppressor functions for these genes. The three genes are composed of multiple exons and map to 2q12.2, 1q25.3, and 5q11.2, respectively. They are well conserved throughout primates, but not other genomes, and were predicted as having no coding potential, although primate-conserved and human-specific short ORFs could be found. Hairpin RNA secondary structures were also predicted. Concluding, this work offers new melanoma-specific genes for future validation as prognostic markers or as targets for the development of therapeutic strategies to treat melanoma.

Predictive factors of non-deterioration of glucose tolerance following a 2-year behavioral intervention

Aim: To identify predictive factors associated with non-deterioration of glucose metabolism following a 2-year behavioral intervention in Japanese-Brazilians. Methods: 295 adults (59.7% women) without diabetes completed 2-year intervention program. Characteristics of those who maintained/improved glucose tolerance status (non-progressors) were compared with those who worsened (progressors) after the intervention. In logistic regression analysis, the condition of non-progressor was used as dependent variable. Results: Baseline characteristics of non-progressors (71.7%) and progressors were similar, except for the former being younger and having higher frequency of disturbed glucose tolerance and lower C-reactive protein (CRP). In logistic regression, non-deterioration of glucose metabolism was associated with disturbed glucose tolerance impaired fasting glucose or impaired glucose tolerance - (p < 0.001) and CRP levels <= 0.04 mg/dL (p = 0.01), adjusted for age and anthropometric variables. Changes in anthropometry and physical activity and achievement of weight and dietary goals after intervention were similar in subsets that worsened or not the glucose tolerance status. Conclusion: The whole sample presented a homogeneous behavior during the intervention. Lower CRP levels and diagnosis of glucose intolerance at baseline were predictors of non-deterioration of the glucose metabolism after a relatively simple intervention, independent of body adiposity.

Decreased fertility in poor responder women is not related to oocyte morphological status

Introduction: In women showing impaired fertility, a decreased response to ovarian stimulation is a major problem, limiting the number of oocytes to be used for assisted reproduction techniques (ART). Despite the several definitions of poor response, it is still a matter of debate whether young poor responder patients also show a decrease in oocyte quality. The objective in this study was to investigate whether poor ovarian response to the superstimulation protocol is accompanied by impaired oocyte quality. Material and methods: This study included 313 patients younger than 35 years old, undergoing intracytoplasmic sperm injection. Patients with four or fewer MII oocytes (poor-responder group, PR, n = 57) were age-matched with normoresponder patients (NR, n = 256). Results: A higher rate of oocyte retrieval and a trend towards an increase in MII oocyte rate were observed in the NR group when compared to the PR group (71.6 +/- 1.1% and 74.1 +/- 1.0% vs. 56.3 +/- 2.9% and 66.5 +/- 3.7%; p < 0.0001 and p = 0.056, respectively). A trend toward increased implantation rates was observed in the NR group when compared to the PR group (44 and 24.5 +/- 2.0% vs. 28.8 and 16.4 +/- 3.9%; p = 0.0305 and p = 0.0651, respectively). Conclusions: Low response to ovarian stimulation is apparently not related to impaired oocyte quality. However, embryos produced from poor responder oocytes show impaired capacity to implant and to carry a pregnancy to term.

Effects of Er:YAG Laser Irradiation on the Microtensile Bond Strength to Bleached Enamel

Objective: The objective of this study was to evaluate the influence of different Er:YAG laser (lambda = 2.94 mu m) energy parameters on the microtensile bond strength (mu TBS) and superficial morphology of bovine enamel bleached with 16% carbamide peroxide. Background: Laser irradiation could improve adhesion to bleached enamel surfaces. Methods: Sixty bovine enamel blocks (7x3x3 mm(3)) were randomly assigned to six groups according to enamel preparation procedures (n = 10): G1-bleaching and Er:YAG laser irradiation with 25.52 J/cm(2) (laser A, LA); G2-bleaching and Er:YAG laser irradiation with 4.42J/cm(2) (laser B, LB); G3-bleaching; G4-Er:YAG laser irradiation with 25.52 J/cm(2); G5-Er:YAG laser irradiation with 4.42J/cm(2); G6-control, no treatment. G1 to G3 were bleached for 6 h during 21 days. Afterwards, enamel surfaces in all groups were slightly abraded with 600-grit SiC papers and G1, G2, G4 and G5 were irradiated according to each protocol. Enamel blocks were then restored with an etch-and-rinse adhesive system and a 4-mm thick composite buildup was made in two increments (n = 9). After 24 h, restored blocks were serially sectioned with a cross-section area of similar to 1 mm(2) at the bonded interface and tested in tension in a universal testing machine (1 mm/min). Failure mode was determined at a magnification of x100 using a stereomicroscope. One treated block of each group was selected for scanning electron microscopy (SEM) analysis. mu TBS data were analyzed by two-way ANOVA and no statistical differences were observed among groups. Results: Mean bond strengths (SD) in MPa were: G1-30.4(6.2); G2-27.9(8.5); G3-32.3(3.9); G4-23.7(5.8); G5-29.3(6.0); G6-29.1(6.1). A large number of adhesive failures was recorded for bleached and irradiated enamel surfaces. Conclusions: Bleached enamel surfaces mu TBS values were not significantly different from those of unbleached enamel. Even though Er:YAG laser irradiation with both parameters had no influence on mu TBS for bleached and unbleached enamel, SEM analysis revealed that Er:YAG laser irradiation with 25.52J/cm(2) should not be recommended, as enamel ablation was observed, whereas irradiation with 4.42J/cm(2) did not promote any remarkable changes on enamel surface.