Claudio De Felice

Erythrocyte caspase-3 activation and oxidative imbalance in erythrocytes and in plasma of type 2 diabetic patients

An increased oxidative stress and a decreased life span of erythrocytes (RBCs) are reported in patients with diabetes. Aim of this study was to assess in RBCs from patients with type 2 diabetes whether downstream effector mechanisms of apoptosis, such as activation of caspase-3, is operative, and whether an iron-related oxidative imbalance, occurring inside RBCs and in plasma, could be involved in caspase-3 activation. In 26 patients with type 2 diabetes and in 12 healthy subjects, oxidative stress was evaluated by means of different markers; non-protein-bound iron, methemoglobin and glutathione were determined in RBCs, and non-protein-bound iron was also determined in plasma. Erythrocyte caspase-3 activation was evaluated by an immunosorbent enzyme assay. Arterial hypertension, demographic and standard biochemical data were also evaluated. The results show, for the first time, that type 2 diabetic RBCs put into motion caspase-3 activation, which is significantly higher than in control RBCs. Such an effector mechanism of “eryptosis” was positively correlated to blood glucose levels and to the increased plasma NPBI level. Caspase-3 activation was also positively correlated to occurrence of arterial hypertension. The results suggest that an extracellular oxidative milieu can be responsible for erythrocyte caspase-3 activation in patients with type 2 diabetes. In turn, caspase-3 activation can be envisaged as a novel mechanism which, by impairing the maintenance of erythrocyte shape and function, might contribute to the shortened life span of RBCs from patients with type 2 diabetes and to hemorheological disorders observed in these patients.

Systemic oxidative stress in classic Rett syndrome

Rett syndrome (RS), a progressive severe neurodevelopmental disorder mainly caused by de novo mutations in the X-chromosomal MeCP2 gene encoding the transcriptional regulator methyl-CpG-binding protein 2, is a leading cause of mental retardation with autistic features in females. However, its pathogenesis remains incompletely understood, and no effective therapy is available to date. We hypothesized that a systemic oxidative stress may play a key role in the pathogenesis of classic RS. Patients with classic RS (n=59) and control subjects (n=43) were evaluated. Oxidative stress markers included intraerythrocyte non-protein-bound iron (NPBI; i.e., free iron), plasma NPBI, F2-isoprostanes (F2-IsoPs, as free, esterified, and total forms), and protein carbonyls. Lung ventilation/perfusion (V/Q) ratio was assessed using a portable gas analyzer, and RS clinical severity was evaluated using standard scales. Significantly increased intraerythrocyte NPBI (2.73-fold), plasma NPBI (x 6.0), free F(2)-IsoP (x1.85), esterified F2-IsoP (x 1.69), total F2-IsoP (x 1.66), and protein carbonyl (x 4.76) concentrations were evident in RS subjects and associated with reduced (-10.53%) arterial oxygen levels compared to controls. Biochemical evidence of oxidative stress was related to clinical phenotype severity and lower peripheral and arterial oxygen levels. Pulmonary V/Q mismatch was found in the majority of the RS population. These data identify hypoxia-induced oxidative stress as a key factor in the pathogenesis of classic RS and suggest new therapeutic approaches based on oxidative stress modulation.

The pulse oximeter perfusion index as a predictor for high illness severity in neonates

Following the first development in its modern form in 1972 by Takuo Aoyagi [1], pulse oximetry has become invaluable for monitoring oxygenation and pulse rate [4]. A constant amount of light (DC) from the pulseoximeter is absorbed by skin, other tissues, and nonpulsatile blood, while a variable amount of light (AC) is absorbed by pulsating arterial inflow. The pulsatile signal indexed against the non-pulsatile signal and expressed as a percentage (AC/DC · 100) is commonly referred to as the perfusion index (PI). Skin colourimetry is related to illness severity in newborns [2] but its utility as a monitoring tool is partially limited by requiring direct caregiver assessment. As local skin vasoconstriction can be associated with skin colour changes, the usefulness of PI for assessing neonatal illness severity was similarly tested. A prospective study was carried out on 101 inborn or outborn Caucasian neonates (52 males, 49 females; gestational age 34.7±4.0 weeks, range 24.7–41.1 weeks; birth weight 2310±950 g, range 410–4170 g) during the first 24 h after admission. Illness severity was determined using the Score for Neonatal Acute Physiology (SNAP) [5] and the infants were categorised as either high or low severity of illness, defined by the presence of severe neonatal morbidity and/or a 24 h SNAP score >10 [2]. PI values were assessed using a Masimo SET Radical pulse-oximeter (Masimo Corp., Irvine, Calif., USA) with the sensor placed randomly on either foot. After the pulse wave was verified to be artifact-free, the PI values were manually captured by an operator, who was unaware of the infant illness severity group, at least every 0.3 min for a duration of 10.6±2.3 min (95% CI 9.9–11.4 min, range: 10–15 min). SpO2, pulse rate, body (skin and core) temperature, and blood pressure were also measured. Out of a total of 2,571 measurements, 2,546 (99.02%) artifact-free readings were obtained. The higher severity group showed a significantly higher frequency of severe neonatal morbidity (P=0.025), as determined by the presence of at least one of the following: sepsis or pneumonia; bronchopulmonary dysplasia; intraventricular haemorrhage grade 3 or more; periventricular leukomalacia grade 3 or more; retinopathy of prematurity grade 3 or more; and necrotising enterocolitis [2]. Predictive accuracy for identifying newborns with higher severity for different cut-off values of PI, SpO2, and pulse rate was calculated using receiver operating characteristic (ROC) curve [3]. Model calibration was evaluated using v to compare the expected values (according to the classification of infants into two severity categories) with the expected values (according to the SpO2, pulse rate and PI values). According to the predefined criteria, 43 neonates (42.6%) were allocated to the high severity group and 58 to the low severity group. Male to female ratio, gestational age, birth weight, body temperature, mean blood pressure, and use of peripheral vasoconstrictors and vasodilators were not significantly different between the two severity groups (P‡0.50). Mean PI values were 1.54±0.80 (range: 0.22–5.22). Significantly lower PI values (0.86±0.26 versus 2.02±0.70; P<0.0001), lower SpO2 (93.3±5.4% versus 95.1±3.9%, P<0.0001) and higher pulse rate Eur J Pediatr (2002) 161: 561–562 DOI 10.1007/s00431-002-1042-5

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.

DI-2-ethylhexyl phthalate and endocrine disruption: a review.

Esters of o-phthalic acid are widely distributed in the ecosystem. The phthalate acid esters (PAEs) are used as plasticizers in enormous quantities for a variety of industrial uses in the formulation of plastics. They are also used as solvents in certain industrial processes and as vehicles for pesticides and have been described as being among the most abundant man-made environmental pollutants. Plasticizers have been shown to elute at a constant rate from plastic products to the environment. Increasing chemical use needs a better understanding of how these pollutants may affect human health. In particular, certain members of this chemical class, such as di-[2-ethylhexyl]-phthalate (DEHP), have been shown to cause reproductive and developmental toxicity and are suspected to be endocrine disruptors.

Small thymus in very low birth weight infants born to mothers with subclinical chorioamnionitis

Chorioamnionitis, a major cause of preterm birth with significant neonatal morbidity and mortality, frequently occurs in mothers who are free of symptoms. A combined clinical, radiologic, and pathologic study of 129 very low birth weight infants indicated a significant association between a markedly decreased thymic size at birth and subclinical chorioamnionitis.

Di-(2-Ethylhexyl) Phthalate and Autism Spectrum Disorders:

ASDs (autism spectrum disorders) are a complex group of neurodevelopment disorders, still poorly understood, steadily rising in frequency and treatment refractory. Extensive research has been so far unable to explain the aetiology of this condition, whereas a growing body of evidence suggests the involvement of environmental factors. Phthalates, given their extensive use and their persistence, are ubiquitous environmental contaminants. They are EDs (endocrine disruptors) suspected to interfere with neurodevelopment. Therefore they represent interesting candidate risk factors for ASD pathogenesis. The aim of this study was to evaluate the levels of the primary and secondary metabolites of DEHP [di-(2-ethylhexyl) phthalate] in children with ASD. A total of 48 children with ASD (male: 36, female: 12; mean age: 11±5 years) and age- and sex-comparable 45 HCs (healthy controls; male: 25, female: 20; mean age: 12±5 years) were enrolled. A diagnostic methodology, based on the determination of urinary concentrations of DEHP metabolites by HPLC-ESI-MS (HPLC electrospray ionization MS), was applied to urine spot samples. MEHP [mono-(2-ethylhexenyl) 1,2-benzenedicarboxylate], 6-OH-MEHP [mono-(2-ethyl-6-hydroxyhexyl) 1,2-benzenedicarboxylate], 5-OH-MEHP [mono-(2-ethyl-5-hydroxyhexyl) 1,2-benzenedicarboxylate] and 5-oxo-MEHP [mono-(2-ethyl-5-oxohexyl) 1,2-benzenedicarboxylate] were measured and compared with unequivocally characterized, pure synthetic compounds (>98%) taken as standard. In ASD patients, significant increase in 5-OH-MEHP (52.1%, median 0.18) and 5-oxo-MEHP (46.0%, median 0.096) urinary concentrations were detected, with a significant positive correlation between 5-OH-MEHP and 5-oxo-MEHP (rs = 0.668, P<0.0001). The fully oxidized form 5-oxo-MEHP showed 91.1% specificity in identifying patients with ASDs. Our findings demonstrate for the first time an association between phthalates exposure and ASDs, thus suggesting a previously unrecognized role for these ubiquitous environmental contaminants in the pathogenesis of autism.

Spleen Depletion in Neonatal Sepsis and Chorioamnionitis

Neonatal sepsis and chorioamnionitis induce morphologic modifications and shrinkage of the thymus. We show fetal and neonatal morphologic modifications of the spleen in the same autopsy subjects as previously used to describe thymus shrinkage, including 10 preterm or full-term neonates who died of proven sepsis within 48 hours after birth and 20 fetuses spontaneously aborted because of extensive ascending chorioamnionitis. Control subjects included 10 fetuses from induced termination of pregnancy and 10 neonates who died suddenly during the perinatal period without evidence of chorioamnionitis. Spleen cell populations were studied by means of immunohistochemical analysis. Neonatal sepsis occurred with severe spleen depletion, involving both B and T lymphocytes (P < .001). Fetuses with chorioamnionitis also showed spleen cell depletion. These observations, to our knowledge not described before, indicate that preterm and term neonates show an inflammatory reaction similar to that of adult patients and that severe chorioamnionitis is associated with a nonspecific inflammatory response comparable to that of sepsis.

New evidences on the altered gut microbiota in autism spectrum disorders

BackgroundAutism spectrum disorders (ASDs) are neurodevelopmental conditions characterized by social and behavioural impairments. In addition to neurological symptoms, ASD subjects frequently suffer from gastrointestinal abnormalities, thus implying a role of the gut microbiota in ASD gastrointestinal pathophysiology.ResultsHere, we characterized the bacterial and fungal gut microbiota in a cohort of autistic individuals demonstrating the presence of an altered microbial community structure. A fraction of 90% of the autistic subjects were classified as severe ASDs. We found a significant increase in the Firmicutes/Bacteroidetes ratio in autistic subjects due to a reduction of the Bacteroidetes relative abundance. At the genus level, we observed a decrease in the relative abundance of Alistipes, Bilophila, Dialister, Parabacteroides, and Veillonella in the ASD cohort, while Collinsella, Corynebacterium, Dorea, and Lactobacillus were significantly increased. Constipation has been then associated with different bacterial patterns in autistic and neurotypical subjects, with constipated autistic individuals characterized by high levels of bacterial taxa belonging to Escherichia/Shigella and Clostridium cluster XVIII. We also observed that the relative abundance of the fungal genus Candida was more than double in the autistic than neurotypical subjects, yet due to a larger dispersion of values, this difference was only partially significant.ConclusionsThe finding that, besides the bacterial gut microbiota, also the gut mycobiota contributes to the alteration of the intestinal microbial community structure in ASDs opens the possibility for new potential intervention strategies aimed at the relief of gastrointestinal symptoms in ASDs.

F2-dihomo-isoprostanes as potential early biomarkers of lipid oxidative damage in Rett syndrome

Oxidative damage has been reported in Rett syndrome (RTT), a pervasive developmental disorder caused in up to 95% of cases by mutations in the X-linked methyl-CpG binding protein 2 gene. Herein, we have synthesized F2-dihomo-isoprostanes (F2-dihomo-IsoPs), peroxidation products from adrenic acid (22:4 n-6), a known component of myelin, and tested the potential value of F2-dihomo-IsoPs as a novel disease marker and its relationship with clinical presentation and disease progression. F2-dihomo-IsoPs were determined by gas chromatography/negative-ion chemical ionization tandem mass spectrometry. Newly synthesized F2-dihomo-IsoP isomers [ent-7(RS)-F2t-dihomo-IsoP and 17-F2t-dihomo-IsoP] were used as reference standards. The measured ions were the product ions at m/z 327 derived from the [M–181]− precursor ions (m/z 597) produced from both the derivatized ent-7(RS)-F2t-dihomo-IsoP and 17-F2t-dihomo-IsoP. Average plasma F2-dihomo-IsoP levels in RTT were about one order of magnitude higher than those in healthy controls, being higher in typical RTT as compared with RTT variants, with a remarkable increase of about two orders of magnitude in patients at the earliest stage of the disease followed by a steady decrease during the natural clinical progression. hese data indicate for the first time that quantification of F2-dihomo-IsoPs in plasma represents an early marker of the disease and may provide a better understanding of the pathogenic mechanisms behind the neurological regression in patients with RTT