Isoprostanoid Plasma Levels Are Relevant to Cerebral Adrenoleukodystrophy Disease.

Cerebral adrenoleukodystrophy (ALD) is a rare neuroinflammatory disorder characterized by progressive demyelination. Mutations within the ABCD1 gene result in very long-chain fatty acid (VLCFA) accumulation within the peroxisome, particularly in the brain. While this VLCFA accumulation is known to be the driving cause of the disease, oxidative stress can be a contributing factor. For patients with early cerebral disease, allogeneic hematopoietic stem cell transplantation (HSCT) is the standard of care, and this can be supported by antioxidants. To evaluate the involvement of fatty acid oxidation in the disease, F2-isoprostanes (F2-IsoPs), F2-dihomo-isoprostanes (F2-dihomo-IsoPs) and F4-neuroprostanes (F4-NeuroPs)-which are oxygenated metabolites of arachidonic (ARA), adrenic (AdA) and docosahexaenoic (DHA) acids, respectively-in plasma samples from ALD subjects (n = 20)-with various phenotypes of the disease-were measured. Three ALD groups were classified according to patients with: (1) confirmed diagnosis of ALD but without cerebral disease; (2) cerebral disease in early period post-HSCT (<100 days post-HSCT) and on intravenous N-acetyl-L-cysteine (NAC) treatment; (3) cerebral disease in late period post-HSCT (beyond 100 days post-HSCT) and off NAC therapy. In our observation, when compared to healthy subjects (n = 29), in ALD (i), F2-IsoPs levels were significantly (p < 0.01) increased in all patients, with the single exception of the early ALD and on NAC subjects; (ii) significant elevated (p < 0.0001) amounts of F2-dihomo-IsoPs were detected, with the exception of patients with a lack of cerebral disease; (iii), a significant increase (p < 0.003) in F4-NeuroP plasma levels was detected in all ALD patients. Moreover, F2-IsoPs plasma levels were significantly higher (p = 0.038) in early ALD in comparison to late ALD stage, and F4-NeuroPs were significantly lower (p = 0.012) in ALD subjects with a lack of cerebral disease in comparison to the late disease stage. Remarkably, plasma amounts of all investigated isoprostanoids were shown to discriminate ALD patients vs. healthy subjects. Altogether, isoprostanoids are relevant to the phenotype of X-ALD and may be helpful in predicting the presence of cerebral disease and establishing the risk of progression.

Antiglycative Activity and RAGE Expression in Rett Syndrome.

Rett syndrome (RTT) is a human neurodevelopmental disorder, whose pathogenesis has been linked to both oxidative stress and subclinical inflammatory status (OxInflammation). Methylglyoxal (MG), a glycolytic by-product with cytotoxic and pro-oxidant power, is the major precursor in vivo of advanced glycation end products (AGEs), which are known to exert their detrimental effect via receptor- (e.g., RAGE) or non-receptor-mediated mechanisms in several neurological diseases. On this basis, we aimed to compare fibroblasts from healthy subjects (CTR) with fibroblasts from RTT patients (N = 6 per group), by evaluating gene/protein expression patterns, and enzymatic activities of glyoxalases (GLOs), along with the levels of MG-dependent damage in both basal and MG-challenged conditions. Our results revealed that RTT is linked to an alteration of the GLOs system (specifically, increased GLO2 activity), that ensures unchanged MG-dependent damage levels. However, RTT cells underwent more pronounced cell death upon exogenous MG-treatment, as compared to CTR, and displayed lower RAGE levels than CTR, with no alterations following MG-treatment, thus suggesting that an adaptive response to dicarbonyl stress may occur. In conclusion, besides OxInflammation, RTT is associated with reshaping of the major defense systems against dicarbonyl stress, along with an altered cellular stress response towards pro-glycating insults.

Cytokines profile and peripheral blood mononuclear cells morphology in Rett and autistic patients.

A potential role for immune dysfunction in autism spectrum disorders (ASD) has been well established. However, immunological features of Rett syndrome (RTT), a genetic neurodevelopmental disorder closely related to autism, have not been well addressed yet. By using multiplex Luminex technology, a panel of 27 cytokines and chemokines was evaluated in serum from 10 RTT patients with confirmed diagnosis of MECP2 mutation (typical RTT), 12 children affected by classic autistic disorder and 8 control subjects. The cytokine/chemokine gene expression was assessed by real time PCR on mRNA of isolated peripheral blood mononuclear cells (PBMCs). Moreover, ultrastructural analysis of PBMCs was performed using transmission electron microscopy (TEM). Significantly higher serum levels of interleukin-8 (IL-8), IL-9, IL-13 were detected in RTT compared to control subjects, and IL-15 shows a trend toward the upregulation in RTT. In addition, IL-1ß and VEGF were the only down-regulated cytokines in autistic patients with respect to RTT. No difference in cytokine/chemokine profile between autistic and control groups was detected. These data were also confirmed by ELISA real time PCR. At the ultrastructural level, the most severe morphological abnormalities were observed in mitochondria of both RTT and autistic PBMCs. In conclusion, our study shows a deregulated cytokine/chemokine profile together with morphologically altered immune cells in RTT. Such abnormalities were not quite as evident in autistic subjects. These findings indicate a possible role of immune dysfunction in RTT making the clinical features of this pathology related also to the immunology aspects, suggesting, therefore, novel possible therapeutic interventions for this disorder.

Exploring the link between scavenger receptor B1 expression and chronic obstructive pulmonary disease pathogenesis.

Chronic obstructive pulmonary disease (COPD) has been recognized as one of the major causes of morbidity and mortality in the United States; it is the third leading cause of deaths in the United States, with approximately 15 million Americans affected with COPD. Although exposure to cigarette smoke has been shown to be the main, if not the only, risk factor for COPD, the mechanisms underlying this association remain unclear. Most smokers do not develop COPD, suggesting that a combination of exposure and susceptibility (genetic background) is required. Several mechanisms contribute to the pathogenesis of COPD, such as influx of inflammatory cells into the lung, imbalance between proteolytic and antiproteolytic molecules, disruption of the balance between apoptosis and replenishment of structural cells in the lung, and disruption of oxidant/antioxidant balance. The scavenger receptor BI (SRB1) plays an important role in mediating the uptake of high-density lipoprotein (HDL)-derived cholesterol and cholesteryl ester in tissues. In addition to its role as the HDL receptor, SRB1 is also involved in pathogen recognition, identification of apoptotic cells, tissue antioxidant uptake (tocopherol and carotenoids), and lung surfactant composition, all factors involved in COPD pathogenesis. Therefore, it is possible that lung SRB1 levels are involved in the development of COPD.

Redox imbalance and morphological changes in skin fibroblasts in typical Rett syndrome.

Evidence of oxidative stress has been reported in the blood of patients with Rett syndrome (RTT), a neurodevelopmental disorder mainly caused by mutations in the gene encoding the Methyl-CpG-binding protein 2. Little is known regarding the redox status in RTT cellular systems and its relationship with the morphological phenotype. In RTT patients (n = 16) we investigated four different oxidative stress markers, F2-Isoprostanes (F2-IsoPs), F4-Neuroprostanes (F4-NeuroPs), nonprotein bound iron (NPBI), and (4-HNE PAs), and glutathione in one of the most accessible cells, that is, skin fibroblasts, and searched for possible changes in cellular/intracellular structure and qualitative modifications of synthesized collagen. Significantly increased F4-NeuroPs (12-folds), F2-IsoPs (7.5-folds) NPBI (2.3-folds), 4-HNE PAs (1.48-folds), and GSSG (1.44-folds) were detected, with significantly decreased GSH (-43.6%) and GSH/GSSG ratio (-3.05 folds). A marked dilation of the rough endoplasmic reticulum cisternae, associated with several cytoplasmic multilamellar bodies, was detectable in RTT fibroblasts. Colocalization of collagen I and collagen III, as well as the percentage of type I collagen as derived by semiquantitative immunofluorescence staining analyses, appears to be significantly reduced in RTT cells. Our findings indicate the presence of a redox imbalance and previously unrecognized morphological skin fibroblast abnormalities in RTT patients.

Inflammatory lung disease in Rett syndrome.

Rett syndrome (RTT) is a pervasive neurodevelopmental disorder mainly linked to mutations in the gene encoding the methyl-CpG-binding protein 2 (MeCP2). Respiratory dysfunction, historically credited to brainstem immaturity, represents a major challenge in RTT. Our aim was to characterize the relationships between pulmonary gas exchange abnormality (GEA), upper airway obstruction, and redox status in patients with typical RTT (n = 228) and to examine lung histology in a Mecp2-null mouse model of the disease. GEA was detectable in ~80% (184/228) of patients versus ~18% of healthy controls, with "high" (39.8%) and "low" (34.8%) patterns dominating over "mixed" (19.6%) and "simple mismatch" (5.9%) types. Increased plasma levels of non-protein-bound iron (NPBI), F2-isoprostanes (F2-IsoPs), intraerythrocyte NPBI (IE-NPBI), and reduced and oxidized glutathione (i.e., GSH and GSSG) were evidenced in RTT with consequently decreased GSH/GSSG ratios. Apnea frequency/severity was positively correlated with IE-NPBI, F2-IsoPs, and GSSG and negatively with GSH/GSSG ratio. A diffuse inflammatory infiltrate of the terminal bronchioles and alveoli was evidenced in half of the examined Mecp2-mutant mice, well fitting with the radiological findings previously observed in RTT patients. Our findings indicate that GEA is a key feature of RTT and that terminal bronchioles are a likely major target of the disease.

Genes related to mitochondrial functions, protein degradation, and chromatin folding are differentially expressed in lymphomonocytes of Rett syndrome patients.

Rett syndrome (RTT) is mainly caused by mutations in the X-linked methyl-CpG binding protein (MeCP2) gene. By binding to methylated promoters on CpG islands, MeCP2 protein is able to modulate several genes and important cellular pathways. Therefore, mutations in MeCP2 can seriously affect the cellular phenotype. Today, the pathways that MeCP2 mutations are able to affect in RTT are not clear yet. The aim of our study was to investigate the gene expression profiles in peripheral blood lymphomonocytes (PBMC) isolated from RTT patients to try to evidence new genes and new pathways that are involved in RTT pathophysiology. LIMMA (Linear Models for MicroArray) and SAM (Significance Analysis of Microarrays) analyses on microarray data from 12 RTT patients and 7 control subjects identified 482 genes modulated in RTT, of which 430 were upregulated and 52 were downregulated. Functional clustering of a total of 146 genes in RTT identified key biological pathways related to mitochondrial function and organization, cellular ubiquitination and proteosome degradation, RNA processing, and chromatin folding. Our microarray data reveal an overexpression of genes involved in ATP synthesis suggesting altered energy requirement that parallels with increased activities of protein degradation. In conclusion, these findings suggest that mitochondrial-ATP-proteasome functions are likely to be involved in RTT clinical features.

Non-protein-bound iron and 4-hydroxynonenal protein adducts in classic autism.

A link between oxidative stress and autism spectrum disorders (ASDs) remains controversial with opposing views on its role in the pathogenesis of the disease. We investigated for the first time the levels of non-protein-bound iron (NPBI), a pro-oxidant factor, and 4-hydroxynonenal protein adducts (4-HNE PAs), as a marker of lipid peroxidation-induced protein damage, in classic autism. Patients with classic autism (n=20, mean age 12.0±6.2years) and healthy controls (n=18, mean age 11.7±6.5years) were examined. Intraerythrocyte and plasma NPBI were measured by high performance liquid chromatography (HPLC), and 4-HNE PAs in erythrocyte membranes and plasma were detected by Western blotting. The antioxidant defences were evaluated as erythrocyte glutathione (GSH) levels using a spectrophotometric assay. Intraerythrocyte and plasma NPBI levels were significantly increased (1.98- and 3.56-folds) in autistic patients, as compared to controls (p=0.0019 and p<0.0001, respectively); likewise, 4-HNE PAs were significantly higher in erythrocyte membranes and in plasma (1.58- and 1.6-folds, respectively) from autistic patients than controls (p=0.0043 and p=0.0001, respectively). Erythrocyte GSH was slightly decreased (-10.34%) in patients compared to controls (p=0.0215). Our findings indicate an impairment of the redox status in classic autism patients, with a consequent imbalance between oxidative stress and antioxidant defences. Increased levels of NPBI could contribute to lipid peroxidation and, consequently, to increased plasma and erythrocyte membranes 4-HNE PAs thus amplifying the oxidative damage, potentially contributing to the autistic phenotype.

Partial rescue of Rett syndrome by ω-3 polyunsaturated fatty acids (PUFAs) oil.

Evidence of enhanced oxidative stress (O.S.) and lipid peroxidation has been reported in patients with Rett syndrome (RTT), a relatively rare neurodevelopmental disorder progressing in 4-stages, and mainly caused by loss-of-function mutations in the methyl-CpG-binding protein 2. No effective therapy for preventing or arresting the neurologic regression in the disease in its various clinical presentations is available. Based on our prior evidence of enhanced O.S. and lipid peroxidation in RTT patients, herein we tested the possible therapeutic effects of ω-3 polyunsaturated fatty acids (ω-3 PUFAs), known antioxidants with multiple effects, on the clinical symptoms and O.S. biomarkers in the earliest stage of RTT. A total of 20 patients in stage I were randomized (n = 10 subjects per arm) to either oral supplementation with ω-3 PUFAs-containing fish oil (DHA: 72.9 ± 8.1 mg/kg b.w./day; EPA: 117.1 ± 13.1 mg/kg b.w./day; total ω-3 PUFAs: 246.0 ± 27.5 mg/kg b.w./day) for 6 months or no treatment. Primary outcomes were potential changes in clinical symptoms, with secondary outcomes including variations for five O.S. markers in plasma and/or erythrocytes (nonprotein bound iron, F(2)-dihomo-isoprostanes, F(3)-isoprostanes, F(4)-neuroprostanes, and F(2)-isoprostanes). A significant reduction in the clinical severity (in particular, motor-related signs, nonverbal communication deficits, and breathing abnormalities) together with a significant decrease in all the examined O.S. markers was observed in the ω-3 PUFAs supplemented patients, whereas no significant changes were evidenced in the untreated group. For the first time, these findings strongly suggest that a dietary intervention in this genetic disease at an early stage of its natural history can lead to a partial clinical and biochemical rescue.

Morphological changes and oxidative damage in Rett Syndrome erythrocytes.

BACKGROUND: Hypoxemia and increased oxidative stress (OS) have been reported in Rett Syndrome (RTT), a genetical neurodevelopmental disorder. Although OS and hypoxemia can lead to red blood cells (RBCs) shape abnormalities, no information on RBCs morphology in RTT exists. Here, RBCs shape was evaluated in RTT patients and healthy subjects as a function of OS markers, blood oxygenation, pulmonary gas exchange, and cardio-respiratory parameters. METHODS: RBCs morphology was evaluated by Scanning Electron Microscopy. Intraerythrocyte and plasma non protein-bound iron (NPBI), esterified F(2)-Isoprostanes (F(2)-IsoPs), 4-HNE protein adducts (4-HNE PAs) were measured. Pulmonary oxygen gradients and PaO(2) were evaluated by gas analyzers and cardiopulmonary variables by pulse oximetry. In RTT patients these parameters were assessed before and after ω-3 polyunsaturated fatty acids (ω-3 PUFAs) administration. RESULTS: Altered RBCs shapes (leptocytes) and increased NPBI were present in RTT, together with increased erythrocyte membrane esterified F(2)-IsoPs and 4-HNE PAs. Abnormal erythrocyte shapes were related to OS markers levels, pulmonary gas exchange, PaO(2) and cardio-respiratory variables. After ω-3 PUFAs, a decrease of leptocytes was accompanied by a progressive increase in reversible forms of RBCs. This partial RBCs morphology rescue was related to decreased OS damage markers, improved pulmonary oxygen exchange, and cardiopulmonary physiology. CONCLUSIONS: These findings indicate that in RTT 1) RBCs shape is altered; 2) the OS-hypoxia diad is critical in generating altered RBCs shape and membrane damage; 3) ω-3 PUFAs are able to partially rescue RBCs morphology and the OS-derived damage. GENERAL SIGNIFICANCE: RBCs morphology is an important biosensor for OS imbalance and chronic hypoxemia.

Syndromic mental retardation with thrombocytopenia due to 21q22.11q22.12 deletion: Report of three patients.

During the last few years, an increasing number of microdeletion/microduplication syndromes have been delineated. This rapid evolution is mainly due to the availability of microarray technology as a routine diagnostic tool. Microdeletions of the 21q22.11q22.12 region encompassing the RUNX1 gene have been reported in nine patients presenting with syndromic thrombocytopenia and mental retardation. RUNX1 gene is responsible for an autosomal dominant platelet disorder with predisposition to acute myelogenous leukemia. We report on three novel patients with an overlapping "de novo" interstitial deletion involving the band 21q22 characterized by array-CGH. All our patients presented with severe developmental delay, dysmorphic features, behavioral problems, and thrombocytopenia. Comparing the clinical features of our patients with the overlapping ones already reported two potential phenotypes related to 21q22 microdeletion including RUNX1 were highlighted: thrombocytopenia with +/- mild dysmorphic features and syndromic thrombocytopenia with growth and developmental delay.