Pathway analysis of glioblastoma tissue after preoperative treatment with the EGFR tyrosine kinase inhibitor gefitinib--a phase II trial.

Amplification of the epidermal growth factor receptor (EGFR) gene is one of the most common oncogenic alterations in glioblastoma (45%) making it a prime target for therapy. However, small molecule inhibitors of the EGFR tyrosine kinase showed disappointing efficacy in clinical trials for glioblastoma. Here we aimed at investigating the molecular effects of the tyrosine kinase inhibitor gefitinib on the EGFR signaling pathway in human glioblastoma. Twenty-two patients selected for reoperation of recurrent glioblastoma were treated within a phase II trial for 5 days with 500 mg gefitinib before surgery followed by postoperative gefitinib until recurrence. Resected glioblastoma tissues exhibited high concentrations of gefitinib (median, 4.1 μg/g), 20 times higher than respective plasma. EGFR-pathway activity was evaluated with phosphorylation-specific assays. The EGFR was efficiently dephosphorylated in treated patients as compared to a control cohort of 12 patients. However, no significant effect on 12 pathway constituents was detected. In contrast, in vitro treatment of a glioblastoma cell line, BS-153, with endogenous EGFRwt amplification and EGFRvIII expression resulted not only in dephosphorylation of the EGFR, but also of key regulators in the pathway such as AKT. Treating established xenografts of the same cell line as an in vivo model showed dephosphorylation of the EGFR without affecting downstream signal transductors, similar to the human glioblastoma. Taken together, gefitinib reaches high concentrations in the tumor tissue and efficiently dephosphorylates its target. However, regulation of downstream signal transducers in the EGFR pathway seems to be dominated by regulatory circuits independent of EGFR phosphorylation.

Benznidazole biotransformation in rat heart microsomal fraction without observable ultrastructural alterations: comparison to Nifurtimox-induced cardiac effects

Benznidazole (Bz) and Nifurtimox (Nfx) have been used to treat Chagas disease. As recent studies have de-monstrated cardiotoxic effects of Nfx, we attempted to determine whether Bz behaves similarly. Bz reached the heart tissue of male rats after intragastric administration. No cytosolic Bz nitroreductases were detected, although microsomal NADPH-dependent Bz nitroreductase activity was observed, and appeared to be mediated by P450 reductase. No ultrastructurally observable deleterious effects of Bz were detected, in contrast to the overt cardiac effects previously reported for Nfx. In conclusion, when these drugs are used in chagasic patients, Bz may pose a lesser risk to heart function than Nfx when any cardiopathy is present.

Alterations of specific biomarkers of metabolic pathways in vascular tree from patients with Type 2 diabetes.

The aims of this study were to check whether different biomarkers of inflammatory, apoptotic, immunological or lipid pathways had altered their expression in the occluded popliteal artery (OPA) compared with the internal mammary artery (IMA) and femoral vein (FV) and to examine whether glycemic control influenced the expression of these genes. The study included 20 patients with advanced atherosclerosis and type 2 diabetes mellitus, 15 of whom had peripheral arterial occlusive disease (PAOD), from whom samples of OPA and FV were collected. PAOD patients were classified based on their HbA1c as well (HbA1c ≤ 6.5) or poorly (HbA1c > 6.5) controlled patients. Controls for arteries without atherosclerosis comprised 5 IMA from patients with ischemic cardiomyopathy (ICM). mRNA, protein expression and histological studies were analyzed in IMA, OPA and FV. After analyzing 46 genes, OPA showed higher expression levels than IMA or FV for genes involved in thrombosis (F3), apoptosis (MMP2, MMP9, TIMP1 and TIM3), lipid metabolism (LRP1 and NDUFA), immune response (TLR2) and monocytes adhesion (CD83). Remarkably, MMP-9 expression was lower in OPA from well-controlled patients. In FV from diabetic patients with HbA1c ≤6.5, gene expression levels of BCL2, CDKN1A, COX2, NDUFA and SREBP2 were higher than in FV from those with HbA1c >6.5. The atherosclerotic process in OPA from diabetic patients was associated with high expression levels of inflammatory, lipid metabolism and apoptotic biomarkers. The degree of glycemic control was associated with gene expression markers of apoptosis, lipid metabolism and antioxidants in FV. However, the effect of glycemic control on pro-atherosclerotic gene expression was very low in arteries with established atherosclerosis.

Inverse relation between FASN expression in human adipose tissue and the insulin resistance level.

BACKGROUND Adipose tissue is a key regulator of energy balance playing an active role in lipid storage and may be a dynamic buffer to control fatty acid flux. Just like PPARgamma, fatty acid synthesis enzymes such as FASN have been implicated in almost all aspects of human metabolic alterations such as obesity, insulin resistance or dyslipemia. The aim of this work is to investigate how FASN and PPARgamma expression in human adipose tissue is related to carbohydrate metabolism dysfunction and obesity. METHODS The study included eighty-seven patients which were classified according to their BMI and to their glycaemia levels in order to study FASN and PPARgamma gene expression levels, anthropometric and biochemical variables. RESULTS The main result of this work is the close relation between FASN expression level and the factors that lead to hyperglycemic state (increased values of glucose levels, HOMA-IR, HbA1c, BMI and triglycerides). The correlation of the enzyme with these parameters is inversely proportional. On the other hand, PPARgamma is not related to carbohydrate metabolism. CONCLUSIONS We can demonstrate that FASN expression is a good candidate to study the pathophysiology of type II diabetes and obesity in humans.

Painful total hip replacement due to sciatic nerve entrapment in scar tissue and lipoma.

Painful total hip replacement remains a challenging problem because of the large amount of possible diagnoses. We report about a 64-year-old female patient who was misdiagnosed during 4 years as psychiatric. She suffered of excruciating left retrotrochanteric pain after the implantation of a cementless total hip replacement and revision because of recurrent hip dislocations. Walking was limited to short distances using two crutches. The work-up at this time included the usual diagnoses and remained unsuccessful. No loosening, infection or malposition of the prosthesis could be found, and she had no neurologic deficits in her operated leg. An MRI was obtained to visualize the retrotrochanteric soft tissues and showed a tight scar surrounding the sciatic nerve, which was also compressed by an adjacent lipoma. Therefore, she was reoperated on to remove the lipoma and the scar tissue around the sciatic nerve. To decrease the risk of recurrent scarring around the sciatic nerve, an adhesion barrier was applied before closure. One year after the operation, the patient has no neurologic deficit, no more pain and is able to walk unlimited distances without crutches. Scar tissue around the sciatic nerve is frequently observed during revision surgery. However, we feel that sciatic nerve entrapment by scar tissue should be a part of the differential diagnosis of painful THR. MRI may be a useful tool to achieve this diagnosis.

Fructose-rich diet-induced abdominal adipose tissue endocrine dysfunction in normal male rats.

We have currently studied the changes induced by administration of a fructose-rich diet (FRD) to normal rats in the mass and the endocrine function of abdominal (omental) adipose tissue (AAT). Rats were fed ad libitum a standard commercial chow and tap water, either alone (control diet, CD) or containing fructose (10%, w/vol) (FRD). Three weeks after treatment, circulating metabolic markers and leptin release from adipocytes of AAT were measured. Plasma free fatty acids (FFAs), leptin, adiponectin, and plasminogen activator inhibitor-1 (PAI-1) levels were significantly higher in FRD than in CD rats. AAT mass was greater in FRD than in CD rats and their adipocytes were larger, they secreted more leptin and showed impaired insulin sensitivity. While leptin mRNA expression increased in AAT from FRD rats, gene expression of insulin receptor substrate, IRS1 and IRS2 was significantly reduced. Our study demonstrates that administration of a FRD significantly affects insulin sensitivity and several AAT endocrine/metabolic functions. These alterations could be part of a network of interacting abnormalities triggered by FRD-induced oxidative stress at the AAT level. In view of the impaired glucose tolerance observed in FRD rats, these alterations could play a key role in both the development of metabolic syndrome (MS) and beta-cell failure.

Segmental copy number variation shapes tissue transcriptomes.

Copy number variation (CNV) is a key source of genetic diversity, but a comprehensive understanding of its phenotypic effect is only beginning to emerge. We have generated a CNV map in wild mice and classical inbred strains. Genome-wide expression data from six major organs show not only that expression of genes within CNVs tend to correlate with copy number changes, but also that CNVs influence the expression of genes in their vicinity, an effect that extends up to half a megabase. Genes within CNVs show lower expression and more specific spatial expression patterns than genes mapping elsewhere. Our analyses reveal differential constraint on copy number changes of genes expressed in different tissues. Dosage alterations of brain-expressed genes are less frequent than those of other genes and are buffered by tighter transcriptional regulation. Our study provides initial evidence that CNVs shape tissue transcriptomes on a global scale.

Renal tissue oxygenation in essential hypertension and chronic kidney disease.

Animal studies suggest that renal tissue hypoxia plays an important role in the development of renal damage in hypertension and renal diseases, yet human data were scarce due to the lack of noninvasive methods. Over the last decade, blood oxygenation level-dependent magnetic resonance imaging (BOLD-MRI), detecting deoxyhemoglobin in hypoxic renal tissue, has become a powerful tool to assess kidney oxygenation noninvasively in humans. This paper provides an overview of BOLD-MRI studies performed in patients suffering from essential hypertension or chronic kidney disease (CKD). In line with animal studies, acute changes in cortical and medullary oxygenation have been observed after the administration of medication (furosemide, blockers of the renin-angiotensin system) or alterations in sodium intake in these patient groups, underlining the important role of renal sodium handling in kidney oxygenation. In contrast, no BOLD-MRI studies have convincingly demonstrated that renal oxygenation is chronically reduced in essential hypertension or in CKD or chronically altered after long-term medication intake. More studies are required to clarify this discrepancy and to further unravel the role of renal oxygenation in the development and progression of essential hypertension and CKD in humans.

A 48-week study of fat molecular alterations in HIV naïve patients starting tenofovir/emtricitabine with lopinavir/ritonavir or efavirenz: Greater deleterious effects of efavirenz.

S’han descrit informes contradictoris sobre els efectes d’Efavirenz (EFV) i lopinavir/ritonavir (LPV/r) al teixit adipós subcutani (SAT). L’objectiu d’aquest estudi era evaluar els efectes moleculars i clínics de LPV/r i EFV, tots dos en combinació amb tenofovir/emtricitabina (TDF/FTC), sobre el SAT dels pacients infectats per VIH sense tractament antirretroviral previ. Després de 48 setmanes de tractament, TDF/FTC més LPV/r va augmentar de forma significativa el greix de les extremitats i els paràmetres lipídics, mentre que TDF/FTC/EFV només va augmentar de forma significativa el colesterol total i LDL. La expressió dels gens implicats en la diferenciació dels adipòcits i dels gens relacionats amb la mitocondria no va canviar de forma significativa en el SAT dels pacients exposats a LPV/r, mentre que Cyt b i els gens relacionats amb la imflamació estaven estimulats de forma significativa en el SAT dels pacients exposats a EFV.

Acid-sensing channels in human bladder: expression, function and alterations during bladder pain syndrome.

Purpose: To examine the possible role of H+-activated acid-sensing ion channels (ASICs) in pain perception we characterized their expression in bladder dome biopsies of Bladder Pain Syndrome (BPS) patients and controls, in cultured human urothelium and in urothelial TEU-2 cells.Materials and Methods: Cold cut biopsies from the bladder dome were obtained in 8 asymptomatic controls and 28 patients with symptoms of BPS. ASIC expression was analyzed by QPCR and immunofluorescence. The channel function was measured by electrophysiology.Results: ASIC1a, ASIC2a and ASIC3 mRNAs were detected in human bladder. Similar amounts of ASIC1a and -3 were detected in detrusor smooth muscle, whereas in urothelium ASIC3 levels were higher than -1a. ASIC2a mRNA levels were lower than either -1a or -3 in both layers. ASIC currents were measured in TEU-2 cells and in primary cultures of human urothelium, and ASIC expression was confirmed by QPCR. Differentiation of TEU-2 cells caused an up-regulation of ASIC2a and ASIC3, and a down-regulation of ASIC1a mRNAs. BPS patients showed an up-regulation of ASIC2a and -3 mRNA, whereas ASIC1a remained unchanged. In contrast, the mRNA levels of TRPV1 were down-regulated during BPS. All differences were statistically significant (p<0.05)Conclusions: Several different ASIC subunits are expressed in human bladder and TEU-2 cells, where their levels are regulated during urothelial differentiation. An up-regulation of ASIC2a and -3 in BPS suggests their involvement in increased pain and hyperalgesia. A down-regulation of TRPV1 mRNA levels might indicate a different regulatory mechanism, controlling its expression in human bladder.

Tumor cell budding and laminin-5 expression in colorectal carcinoma can be modulated by the tissue micro-environment.

Expression of laminin-5 alpha3, beta3 and gamma2 protein subunits was investigated in colorectal adenocarcinomas using immunostaining and confocal microscopy. The laminin-5 heterotrimer was found in basement membranes and as extracellular deposits in tumor stroma. In contrast to the alpha3 subunit, which was under-expressed, the gamma2 and beta3 subunits were detected in the cytoplasm of carcinoma cells dissociating (budding) from neoplastic tubules, suggestive of focal alterations in laminin-5 assembly and secretion. Laminin-5 gamma2 or beta3 subunit-reactive budding carcinoma cells expressed cytokeratins but not vimentin; they did not proliferate and were not apoptotic. Furthermore, expression of laminin-5 gamma2 and beta3 subunits in budding cells was associated with focal under-expression of the E-cadherin-beta-catenin complex. Results from xenograft experiments showed that budding activity in colorectal adenocarcinomas could be suppressed when these tumors grew at ectopic s.c. sites in nude mice. In vitro, cultured colon carcinoma cells, but not adenoma-derived tumor cells, shared the laminin-5 phenotype expressed by carcinoma cells in vivo. Using colon carcinoma cell lines implanted orthotopically and invading the cecum of nude mice, the laminin-5-associated budding was restored, indicating that this phenotype is not only determined by tumor cell properties but also dependent on the tissue micro-environment. Our results indicate that both laminin-5 alpha3 subunit expression and cell-cell cohesiveness are altered in budding carcinoma cells, which we consider to be actively invading. We propose that the local tissue micro-environment contributes to these events.

Alterations in the local myocardial motion pattern in patients suffering from pressure overload due to aortic stenosis.

BACKGROUND: MR tissue tagging allows the noninvasive assessment of the locally and temporally resolved motion pattern of the left ventricle. Alterations in cardiac torsion and diastolic relaxation of the left ventricle were studied in patients with aortic stenosis and were compared with those of healthy control subjects and championship rowers with physiological volume-overload hypertrophy. METHODS AND RESULTS: Twelve aortic stenosis patients, 11 healthy control subjects with normal left ventricular function, and 11 world-championship rowers were investigated for systolic and diastolic heart wall motion on a basal and an apical level of the myocardium. Systolic torsion and untwisting during diastole were examined by use of a novel tagging technique (CSPAMM) that provides access to systolic and diastolic motion data. In the healthy heart, the left ventricle performs a systolic wringing motion, with a counterclockwise rotation at the apex and a clockwise rotation at the base. Apical untwisting precedes diastolic filling. In the athlete's heart, torsion and untwisting remain unchanged compared with those of the control subjects. In aortic stenosis patients, torsion is significantly increased and diastolic apical untwisting is prolonged compared with those of control subjects or athletes. CONCLUSIONS: Torsional behavior as observed in pressure- and volume-overloaded hearts is consistent with current theoretical findings. A delayed diastolic untwisting in the pressure-overloaded hearts of the patients may contribute to a tendency toward diastolic dysfunction.

Integrative molecular bioinformatics study of human adrenocortical tumors : microRNA, tissue-specific target prediction, and pathway analysis.

MicroRNAs (miRs) are involved in the pathogenesis of several neoplasms; however, there are no data on their expression patterns and possible roles in adrenocortical tumors. Our objective was to study adrenocortical tumors by an integrative bioinformatics analysis involving miR and transcriptomics profiling, pathway analysis, and a novel, tissue-specific miR target prediction approach. Thirty-six tissue samples including normal adrenocortical tissues, benign adenomas, and adrenocortical carcinomas (ACC) were studied by simultaneous miR and mRNA profiling. A novel data-processing software was used to identify all predicted miR-mRNA interactions retrieved from PicTar, TargetScan, and miRBase. Tissue-specific target prediction was achieved by filtering out mRNAs with undetectable expression and searching for mRNA targets with inverse expression alterations as their regulatory miRs. Target sets and significant microarray data were subjected to Ingenuity Pathway Analysis. Six miRs with significantly different expression were found. miR-184 and miR-503 showed significantly higher, whereas miR-511 and miR-214 showed significantly lower expression in ACCs than in other groups. Expression of miR-210 was significantly lower in cortisol-secreting adenomas than in ACCs. By calculating the difference between dCT(miR-511) and dCT(miR-503) (delta cycle threshold), ACCs could be distinguished from benign adenomas with high sensitivity and specificity. Pathway analysis revealed the possible involvement of G2/M checkpoint damage in ACC pathogenesis. To our knowledge, this is the first report describing miR expression patterns and pathway analysis in sporadic adrenocortical tumors. miR biomarkers may be helpful for the diagnosis of adrenocortical malignancy. This tissue-specific target prediction approach may be used in other tumors too.

Alterations of the 5'untranslated region of SLC16A12 lead to age-related cataract.

PURPOSE. Knowledge of genetic factors predisposing to age-related cataract is very limited. The aim of this study was to identify DNA sequences that either lead to or predispose for this disease. METHODS. The candidate gene SLC16A12, which encodes a solute carrier of the monocarboxylate transporter family, was sequenced in 484 patients with cataract (134 with juvenile cataract, 350 with age-related cataract) and 190 control subjects. Expression studies included luciferase reporter assay and RT-PCR experiments. RESULTS. One patient with age-related cataract showed a novel heterozygous mutation (c.-17A>G) in the 5'untranslated region (5'UTR). This mutation is in cis with the minor G-allele of the single nucleotide polymorphism (SNP) rs3740030 (c.-42T/G), also within the 5'UTR. Using a luciferase reporter assay system, a construct with the patient's haplotype caused a significant upregulation of luciferase activity. In comparison, the SNP G-allele alone promoted less activity, but that amount was still significantly higher than the amount of the common T-allele. Analysis of SLC16A12 transcripts in surrogate tissue demonstrated striking allele-specific differences causing 5'UTR heterogeneity with respect to sequence and quantity. These differences in gene expression were mirrored in an allele-specific predisposition to age-related cataract, as determined in a Swiss population (odds ratio approximately 2.2; confidence intervals, 1.23-4.3). CONCLUSIONS. The monocarboxylate transporter SLC16A12 may contribute to age-related cataract. Sequences within the 5'UTR modulate translational efficiency with pathogenic consequences.

Double incretin receptor knock-out (DIRKO) mice present with alterations of trabecular and cortical micromorphology and bone strength.

A role for gut hormone in bone physiology has been suspected. We evidenced alterations of microstructural morphology (trabecular and cortical) and bone strength (both at the whole-bone - and tissue-level) in double incretin receptor knock-out (DIRKO) mice as compared to wild-type littermates. These results support a role for gut hormones in bone physiology. INTRODUCTION: The two incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), have been shown to control bone remodeling and strength. However, lessons from single incretin receptor knock-out mice highlighted a compensatory mechanism induced by elevated sensitivity to the other gut hormone. As such, it is unclear whether the bone alterations observed in GIP or GLP-1 receptor deficient animals resulted from the lack of a functional gut hormone receptor, or by higher sensitivity for the other gut hormone. The aims of the present study were to investigate the bone microstructural morphology, as well as bone tissue properties, in double incretin receptor knock-out (DIRKO) mice. METHODS: Twenty-six-week-old DIRKO mice were age- and sex-matched with wild-type (WT) littermates. Bone microstructural morphology was assessed at the femur by microCT and quantitative X-ray imaging, while tissue properties were investigated by quantitative backscattered electron imaging and Fourier-transformed infrared microscopy. Bone mechanical response was assessed at the whole-bone- and tissue-level by 3-point bending and nanoindentation, respectively. RESULTS: As compared to WT animals, DIRKO mice presented significant augmentations in trabecular bone mass and trabecular number whereas bone outer diameter, cortical thickness, and cortical area were reduced. At the whole-bone-level, yield stress, ultimate stress, and post-yield work to fracture were significantly reduced in DIRKO animals. At the tissue-level, only collagen maturity was reduced by 9 % in DIRKO mice leading to reductions in maximum load, hardness, and dissipated energy. CONCLUSIONS: This study demonstrated the critical role of gut hormones in controlling bone microstructural morphology and tissue properties.