Brain gliomas and growth factors: immunohistochemical, immunofluorescence, flow cytometry and RT-PCR profile in pediatric age.

Gliomas represent over 50% of tumors occurring in children. Evidence suggests that glioma stem cells (GSCs), maintained by the transforming growth factor-beta (TGF-ß1) pathway, and vascularization substantially contribute to tumor aggressiveness. The identification of important angiogenic factors such as vascular endothelial growth factor (VEGF) may represent a crucial step in the therapeutic approach against tumor growth and metastatic diffusion. The aim of this study was to identify the expression of TGF-ß1, VEGF and VEGF-receptors in brain gliomas. Specimens of 16 gliomas and 4 controls from children aged 0.2-14 years were used in the study. Immunohistochemical analysis and gene expression study from specimens was performed. Flow cytometry analysis on GSCs was performed to ascertain the expression of VEGF and VEGF-R2 in the tumor stem cell compartment. Newly diagnosed gliomas mainly showed moderate to strong VEGF immunostaining and increased expression of pro-inflammatory molecules in glioma cells. The proportion of TGF-ß1 positive endothelial cells was markedly lower in normal brain vessels compared to tumor vessels. These findings demonstrate that the glioma mass is constituted by a phenotypically immature anoxic central area with a proliferating hypoxic layer; the peripheral area is characterized by cell types with a higher degree of differentiation expressing pro-angiogenic factors. Our data have proven that GSCs play a central role in promoting glioma neovascularization. These findings are useful to understand glioma vascularization, have relevant implications in the therapeutic options and may favor new insights into stem cells biology and suggest therapeutic opportunities for the anti-vascular treatment strategy.

Anteriorly placed tumors to the conus: removal by interradicular window.

Tumors anteriorly situated to the medullary conus are rarely encountered and represent a true surgical challenge. We examined the literature on this topic, concluding that there are no previous reports on alternative surgical techniques different to the traditional one. We report two cases of intradural extramedullary tumor operated on by a technique performed through a window opened between the spinal roots, which allows an easy, effective and useful resection. We describe a new operative technique which ensures a complete removal of these tumors and discuss clinical implications in the light of the available literature on this topic.

D’Andrea’s disease (angiomegaly): a currently well-defined nosological entitys.

In 1997 D’Andrea et al. described a new nosological entity the characteristics of which consisted of lengthening, dilation and tortuosity of blood vessels, arteries or veins, less prominent, but also less circumscribed than an aneurysm. This condition does not necessarily imply specific aneurysm formation although aneurysms at multiple sites are a frequent observation. The term used by authors for angiomegaly of the venous system was venomegaly and the analogous condition of the arterial system was termed arteriomegaly. Although tortuosity and dilation of arteries and veins have been widely reported, suggesting a systemic disorder which affects the structural integrity of all vessels, most papers dealing with this intriguing condition did not describe any alterations in the components of vessel walls. In the present paper, the authors describe a well-defined condition, D’Andrea’s Disease (or DD, in this article), analyzing its salient morphological and clinical features and clarifying this pathological condition as a distinct and now well-defined nosological entity.

HLA-dependent autoantibodies against post-translationally modified collagen type II in type 1 diabetes mellitus.

AIMS/HYPOTHESIS: In this study the involvement of oxidative stress in type 1 diabetes mellitus autoimmunity and the possible association with rheumatoid arthritis (RA) was investigated. We tested the hypothesis that oxidative stress induced by chronic hyperglycaemia triggers post-translational modifications and thus the formation of neo-antigens in type 1 diabetes, similar to the ones found in RA. METHODS: Collagen type II (CII), a known autoantigen in RA, was treated with ribose and various reactive oxygen species (ROS). Levels of antibodies specific to native and ROS-modified CII (ROS-CII) were compared in type 1 diabetes, type 2 diabetes and healthy controls, and related to the HLA genotype. RESULTS: Significantly higher binding to ROS-CII vs native CII was observed in type 1 diabetic patients possessing the HLA-DRB1*04 allele irrespective of variables of glucose control (blood glucose or HbA(1c)). Type 1 diabetic patients carrying a DRB1*04 allele with the shared epitope showed the highest risk for ROS-CII autoimmunity, while the DRB1*0301 allele was protective. Conversely, native CII autoimmunity was not associated with any specific DRB1 allele. Positive and inverse seroconversion rates of response to ROS-CII were high in DRB1*04-positive type 1 diabetic patients. CONCLUSION: Hyperglycaemia and oxidative stress may trigger genetically controlled autoimmunity to ROS-CII and may explain the association between type 1 diabetes mellitus and RA.

Association of TCF7L2 gene variants with low GAD autoantibody titre in LADA subjects (NIRAD Study 5).

AIMS: We previously demonstrated the presence of two different populations among adult-onset autoimmune diabetes (latent autoimmume diabetes of adults; LADA) having high or low titre of antibodies to glutamic acid decarboxylase (GADA). The transcription factor 7-like 2 (TCF7L2) gene has been recognized as the major gene associated with Type 2 diabetes. The aim of the present study was to evaluate whether the phenotypic heterogeneity of LADA based on GADA titre is associated with TCF7L2 polymorphisms. METHODS: Two hundred and fifty patients identified as LADA, divided into two subgroups with low (< or = 32 arbitrary units) or high (> 32 units) GADA titre, 620 subjects with Type 2 diabetes [from the Non-Insulin Requiring Autoimmune Diabetes (NIRAD) study cohort of 5330 subjects] in addition to 551 consecutive cases of Type 1 diabetes and 545 normoglycaemic subjects were analysed for the rs12255372 and rs7903146 polymorphisms of the TCF7L2 gene using Taqman. RESULTS: The genotype and allele distributions of the two polymorphisms revealed similar frequencies in subjects with low GADA titre and Type 2 diabetes. High GADA titre, Type 1 diabetes and controls also showed comparable frequencies. A significant increase of GT/TT genotypes of the rs12255372 single-nucleotide polymorphism (SNP) and CT/TT genotypes of the rs7903146 SNP was observed in low GADA titre and Type 2 diabetes compared with high GADA titre, Type 1 diabetes and controls (P < or = 0.04 for both comparisons). The risk alleles of both variants were increased in low GADA titre and Type 2 diabetes compared with high GADA titre, Type 1 diabetes and control subjects (P < 0.02 for all comparisons). CONCLUSIONS: TCF7L2 common genetic variants of susceptibility are associated only with low GADA antibody titre in LADA patients.

A new variation in the promoter region, the -604 C>T, and the Leu72Met polymorphism of the ghrelin gene are associated with protection to insulin resistance.

OBJECTIVE: Previous studies suggested that polymorphisms in the coding region of the preproghrelin were involved in the etiology of obesity and might modulate glucose-induced insulin secretion. We evaluated the association of a new variation, -604C>T, in the promoter region of the ghrelin gene, of Leu72Met (247C>A) and of Gln90Leu (265A>T), all haplotype-tagging single nucleotide polymorphisms (SNPs), with measures of insulin sensitivity in 1420 adult individuals. RESEARCH METHODS: The three SNPs were genotyped using ABI PRISM 7900 HT Sequence Detection System. We used multiple linear regression analysis for quantitative traits and THESIAS software for haplotype analysis. RESULTS: We observed a protective effect exerted by Met72 variant of Leu72Met SNP on insulin resistance parameters; a significant decreasing trend from Leu/Leu to Leu/Met and to Met/Met homozygous subjects in triglycerides, fasting insulin levels and HOMA-IR index (P=0.02, 0.01 and 0.003, respectively), and, consistently, an increase in ghrelin levels (P=0.003) was found. A significant decrease from CC to TC and to TT genotypes in insulin levels and HOMA-IR index was also detected (P=0.00l for both), but only in subjects homozygous for Leu72, where the protective effect of Met72 was not present. The haplotype analysis results supported the data obtained by the evaluation of each single SNP, showing the highest value of insulin levels and HOMA-IR index in the -604(c)247(c) haplotype intermediate value in -604(T)247(C) and lowest value in -604(C)247(A). CONCLUSION: Our observations suggest a protective role of the Met72 variant and of -604 T allele in modulating insulin resistance. These SNPs or an unknown functional variant in linkage disequilibrium could increase ghrelin levels and probably insulin sensitivity.

Low-risk HLA genotype in Type 1 diabetes is associated with less destruction of pancreatic B-cells 12 months after diagnosis.

AIMS: The role of human leukocyte antigen (HLA) genes in the susceptibility to Type 1 diabetes (T1DM) is well known. However, we do not know whether the degree of pancreatic B-cell destruction depends on different HLA genetic risk. The aim of this study was to analyse the influence of DRB1* and DQB1* genes on the rate of pancreatic B-cell loss in a prospective series of 120 consecutive newly diagnosed T1DM subjects in the first 12 months after diagnosis. METHODS: Patients were typed for HLA-DRB1* and DQB1* loci by a reverse line blot assay using an array of immobilized sequence-specific oligonucleotide probes. C-peptide, insulin requirement and glycated haemoglobin (HbA(1c)) were determined at diagnosis and every 3 months for 12 months. The variance of C-peptide as evidence of B-cell loss during follow-up was analysed using the general linear model for repeated-measures procedure. RESULTS: Fasting C-peptide in T1DM subjects with low HLA genetic risk was significantly higher when compared with subjects with moderate or high HLA genetic risk from time of diagnosis up to 12 months (P = 0.007 and P = 0.0002, respectively). Nonetheless, the changes in C-peptide levels over a 12-month period did not differ significantly between T1DM subjects with different HLA genetic risks. CONCLUSIONS: Low-risk HLA genotype in T1DM is associated with less destruction of pancreatic B-cells up to 12 months after diagnosis. These results are useful when designing trials for therapies aimed to prevent the progression of B-cell destruction in recent-onset T1DM.

The glucose clamp reveals an association between adiponectin gene polymorphisms and insulin sensitivity in obese subjects.

Results concerning the association of adiponectin gene polymorphisms (single-nucleotide polymorphisms, SNPs) with obesity, type 2 diabetes (T2DM), metabolic disorders and insulin resistance have not lead to definite conclusions. The aim of our study was to investigate a possible association between the -11391G>A and -11377C>G SNPs of adiponectin gene and measure of insulin sensitivity evaluated by the hyperinsulinemic-euglycemic clamp in a group of 'uncomplicated' obese subjects (with no associated comorbidities) (n=99, mean age 35 years) with a history of obesity lasting at least 10 years. The study of uncomplicated obese subjects, free of possible confounding factors that could interfere with insulin sensitivity, such as pharmacological treatment, provides a good model to assess insulin sensitivity per se. We observed that subjects homozygous for the G allele at locus -11391 had lower M (mg/kg min)/fat-free mass (FFM) index and adiponectin levels compared to subjects with GA+AA genotypes (P=0.002 and P=0.03, respectively) and subjects carrying the -11377G variant had lower M (mg/kg min)/FFM index and adiponectin levels compared to noncarriers (P=0.003 and P=0.03, respectively). Our results imply that the two promoter SNPs, -11391G>A and -11377C>G, of the adiponectin gene are associated with a reduced insulin sensitivity evaluated by hyperinsulinemic-euglycemic clamp in obese subjects.

Residual insulin secretion at diagnosis of type 1 diabetes is independently associated with both, age of onset and HLA genotype.

BACKGROUND: We investigated whether residual insulin secretion and metabolic derangement at diagnosis of type 1 diabetes (T1DM) are influenced by human leukocyte antigens (HLA) class II genes. METHODS: Eight hundred and seventy-one T1DM consecutive Caucasian patients were typed for HLA class II genes. In 300 of these patients, glycated haemoglobin, insulin requirement, baseline C-peptide and body mass index (BMI) Z-score were measured at clinical diagnosis. The effect of the HLA genotypes on the quantitative variables was investigated using multiple linear regression. The beta coefficient regression of the age at onset and HLA genotypes were standardized to compare their specific importance for C-peptide levels. RESULTS: The HLA genotypes were divided in high-, moderate- and low-risk categories. The frequency of high-risk genotype, DRB1*03-DQB1*0201/DRB1*04-DQB1*0302, decreased with increasing age of onset (p < 0.0001, chi(2) linear trend). The presence of the high-risk genotype was independently associated with lower C-peptide levels at diagnosis (p = 0.002). In the regression analysis of C-peptide levels, the standardized beta coefficient for age of onset and high risk compared to low-risk genotypes showed similar results (0.27 and 0.24 respectively). There was a positive association between age of onset and C-peptide (p < 0.0001) and a negative association between age of onset and insulin requirement (p < 0.0001). CONCLUSIONS: The degree of beta-cell destruction at diagnosis of T1DM is independently associated with both, age of onset and HLA genotypes, the two variables exert a similar quantitative effect on residual beta-cell function at diagnosis.