Cristiano Rizzo

Prevalence and prognostic value of quantified electroencephalogram (EEG) alterations in cirrhotic patients

BACKGROUND/AIMS The electroencephalogram (EEG) is frequently altered in cirrhotic patients. We, therefore, performed a study to ascertain the features and the prognosis of cirrhotic patients without current overt hepatic encephalopathy (OHE) who have EEG alterations. METHODS A series of 296 consecutive cirrhotic patients who had undergone quantified-EEG was studied. The median follow-up was 442 days, 128 patients had bouts of OHE and 78 patients died from liver-related causes. Another group of 124 cirrhotic patients with a median follow-up of 223 days was examined to validate the prognostic model. RESULTS EEG alterations were detected in 38% of the patients. The prevalence of EEG alterations was associated with the severity of cirrhosis (class B: odds ratio (OR) = 2.3, 95% confidence interval (CI) = 1.2-4.7; class C: OR = 3.5, 95% CI = 1.6-7.7), but not with the aetiology (alcoholic vs. non-alcoholic: OR = 0.9; 95% CI = 0.5-1.5). The EEG predicted the occurrence of OHE (chi2 = 26; P < 0.001) and mortality (chi2 = 34; P < 0.001), also adjusting for Child-Pugh class by a multivariate analysis. In the patients with a Child-Pugh score of > or = 8, the EEG discriminated between those patients with a higher 1-year risk of OHE (hazard ratio (HR) = 3.3, 95% CI = 1.8-6.1) and death (HR = 3.1, 95% CI = 1.7-5.6). CONCLUSIONS In conclusion, quantified-EEG had a prognostic value for the occurrence of bouts of OHE and mortality in cirrhotic patients.

Extracorporeal dialysis in neonatal hyperammonemia: modalities and prognostic indicators.

Abstract. We investigated the prognostic indicators in ten hyperammonemic neonates: four treated by continuous arteriovenous hemodialysis (CAVHD), four with continuous venovenous hemodialysis (CVVHD), and two with hemodialysis (HD). Plasma ammonium levels decreased significantly within the first 24 h irrespective of dialysis modality (from 1419 to 114 µmol/l, median values; P<0.0001). CVVHD achieved the highest ammonium clearance. HD provided highest ammonium extraction but clearance was hampered by severe hemodynamic instability. Five patients had a good outcome (normal at follow-up of 9–59 months), five had poor outcome (four died and one has severe neurological damage). Total coma duration was shorter in patients who had a good outcome (47±11 vs 78±13 h; P=0.02). Remarkably, only coma duration before dialysis determined this difference (22.2±10.1 vs 48.8±11.2 h; P=0.02). In cases with good outcome, coma duration was <33 h, whereas the others exceeded this limit. The prognosis was not related to dialysis modality, rapidity in reducing ammonium levels or to the underlying metabolic defect. In conclusion, results showed CVVHD to be the optimal modality for extracorporeal ammonium detoxification. However, the most relevant indicator for prognosis was coma duration before the start of dialysis. Therefore, major efforts should be made to refer patients quickly to highly specialized centers.

Spectrum of MMACHC mutations in Italian and Portuguese patients with combined methylmalonic aciduria and homocystinuria, cblC type.

Methylmalonic aciduria (MMA) and homocystinuria, cblC type (MIM 277400) is the most frequent inborn error of vitamin B(12). The recent identification of the disease gene, MMACHC, has permitted preliminary genotype-phenotype correlations. We studied 24 Italian and 17 Portuguese patients with cblC defect to illustrate the spectrum of mutations in a southern European population and discuss the impact that mutation identification has on routine diagnostic procedures. Since the metabolic defect raises the serum levels of homocysteine, we also tested if variants in MTHFR-playing a key role in homocysteine remethylation pathway-could act as genetic modifier in cblC defect. We found that the c.271dupA (accounting for 55% of the MMACH alleles in our cohort) followed by c.394C>T (16%) and c.331C>T (9%) were the most frequent mutations. In our study we also identified a novel mutation (c.544T>C). On the other hand, the MTHFR genotype did not appear to influence age at onset, the clinical phenotype and outcome of patients with cblC defect. This study shows that mutation screening for the most common MMACH mutations occurring in early-onset forms (c.271dupA and c.331C>T) seems to have a high diagnostic yield in a southern European population with cblC defect. Although the identification of the gene defect per se does not predict completely time and severity of disease appearance, our data corroborate the importance of a molecular testing to offer accurate prenatal diagnosis to couples at high risk of having affected children.

Study on the Sternberg Paradigm in Cirrhotic Patients Without Overt Hepatic Encephalopathy

Memory dysfunction is reported in cirrhotics. The aim of this paper was to increase insight into memory function of cirrhotic patients without overt hepatic encephalopathy. Eighty-six consecutive cirrhotics without overt hepatic encephalopathy (aged 54±10 yr., mean±s.d.) and 28 controls (52±10 yr.) with comparable education level were enrolled. Seventeen patients were class A, 55 class B, 14 class C according to Child-Pugh classification; 29 had alcoholic cirrhosis. The presence of subclinical signs of central nervous system dysfunction were assessed by Number Connection Test (NCT) and quantified EEG analysis. Memory scanning was evaluated by reaction times (RTs) in the Sternberg paradigm. MANOVA analysis showed that RTs were higher (F1,99=11, p<0.01) and time outs (TOs) more frequent (F1,110=10, p<0.01) in cirrhotics than in controls, whereas button press errors (BPEs) did not differ significantly (F1,110=2, p=n.s.). In cirrhotics, an interaction Child-Pugh class x memory set size was found (F2,146=4, p<0.05), showing exceedingly delayed RTs with greater memory set size in class C patients. Patients with altered NCT had significantly prolonged RTs (F1,71=4, p<0.05) and more TOs (F1,82=11, p<0.01) than patients with normal NCT. Cirrhotics with altered EEG had significantly prolonged RTs (F2,70=6, p<0.01). RTs were found to be correlated to alpha relative power (r=−0.4, p<0.01) and theta relative power (r=0.4, p<0.01). In conclusion, cirrhotics without over encephalopathy, but with NCT or EEG alterations, perform a computerized digit recognition task more slowly and with higher TOs than cirrhotic patients with normal NCT or EEG. In severe liver insufficiency (class C cirrhotics) also an impairment of memory scanning was detected. Sternberg test performance correlates with NCT and quantitative EEG parameters.

Clinical and molecular findings in hyperornithinemia-hyperammonemia-homocitrullinuria syndrome

The authors report the clinical and molecular findings in eight patients with hyperornithinemia, hyperammonemia, and homocitrullinuria (HHH) syndrome. The most consistent neurologic finding was spastic paraparesis, seen in five of the eight patients. However, all showed signs of pyramidal tract involvement. A broad spectrum of pathogenetic mutations (including missense, nonsense, splice site, insertion, and deletions) were identified in the ORNT1 gene.

Tyrosine hydroxylase deficiency with severe clinical course : clinical and biochemical investigations and optimization of therapy

Tyrosine hydroxylase deficiency was diagnosed after determination of cerebrospinal fluid neurotransmitters and DNA analysis in a child with severe axial hypotonia and hypokinesia associated with dystonic and ballistic movements. L-dopa therapy was unsuccessful, whereas a combination with selegiline, a selective monoamine oxidase-beta inhibitor, with low-dose L-dopa markedly improved the severe clinical picture.

Differential effects of extracellular vesicles secreted by mesenchymal stem cells from different sources on glioblastoma cells

Background: Malignant glial tumors, including glioblastoma multiforme, account for 15 – 20% of pediatric CNS malignancies. They are most resistant to therapy and are associated with a poor prognosis. Objective: Given the ability of mesenchymal stem cells (MSCs) to affect glioma growth, we investigated the effects of extracellular vesicles (EVs) derived from MSCs on U87MG glioblastoma cells line. Methods: EVs were isolated from culture media of MSCs from different sources, including bone marrow (BM), umbilical cord (UC) and adipose tissue (AT) and added to U87MG culture. The internalization and the effects of BM-, UC- and AT-MSC-EVs on proliferation and apoptosis of tumor cells were evaluated. Results: Both confocal microscopy and FACS analysis showed internalization of EVs into tumor cells. BM- and UC-MSC-EVs decreased cell proliferation, while an opposite effect was observed with AT-MSC-EVs. Moreover, both BM- and UC-MSC-EVs induced apoptosis of glioblastoma cells, while AT-MSC-EVs had no effect. Loading UC-MSC-EVs with Vincristine further increased cytotoxicity when compared both to the free drug and to untreated EVs. Conclusions: Different effects of MSC-EVs on cancer cells were observed depending on their tissue of origin. Moreover, MSC-EVs can deliver antiblastic drugs to glioblastoma cells.

Evidence for genetic heterogeneity in D-2-hydroxyglutaric aciduria†

We performed molecular, enzyme, and metabolic studies in 50 patients with D‐2‐hydroxyglutaric aciduria (D‐2‐HGA) who accumulated D‐2‐hydroxyglutarate (D‐2‐HG) in physiological fluids. Presumed pathogenic mutations were detected in 24 of 50 patients in the D‐2‐hydroxyglutarate dehydrogenase (D2HGDH) gene, which encodes D‐2‐hydroxyglutarate dehydrogenase (D‐2‐HGDH). Enzyme assay of D‐2‐HGDH confirmed that all patients with mutations had impaired enzyme activity, whereas patients with D‐2‐HGA whose enzyme activity was normal did not have mutations. Significantly lower D‐2‐HG concentrations in body fluids were observed in mutation‐positive D‐2‐HGA patients than in mutation‐negative patients. These results imply that multiple genetic loci may be associated with hyperexcretion of D‐2‐HG. Accordingly, we suggest a new classification: D‐2‐HGA Type I associates with D‐2‐HGDH deficiency, whereas idiopathic D‐2‐HGA manifests with normal D‐2‐HGDH activity and higher D‐2‐HG levels in body fluids compared with Type I patients. It remains possible that several classifications for idiopathic D‐2‐HGA patients with diverse genetic loci will be revealed in future studies. Hum Mutat 31:1–5, 2010.

Characteristic Acylcarnitine Profiles in Inherited Defects of Peroxisome Biogenesis: A Novel Tool for Screening Diagnosis Using Tandem Mass Spectrometry

Patients with inherited defects of peroxisomal metabolism, a class of diseases with marked clinical and genetic heterogeneity, show a characteristic phenotype in most cases with severe neurologic impairment, craniofacial abnormalities, and hepatic and kidney dysfunction. For the differential diagnosis of clinically suspected cases, a complex biochemical and genetic approach is required. Analysis of plasma very-long-chain fatty acids is a reliable screening method to detect most but not all peroxisomal disorders. To study the potential presence of abnormal acylcarnitine species in plasma and blood, we screened by tandem mass spectrometry a series of patients affected by a peroxisome biogenesis disorder (PBD) and compared the results with those obtained in patients with isolated peroxisomal defects (e.g. D-bifunctional protein deficiency, X-linked adrenoleukodystrophy) and mitochondrial long-chain fatty acid oxidation defects. The most relevant finding observed in plasma of patients with PBD was a significant increase of long-chain dicarboxylic C16- and C18-carnitine, i.e. hexadecanedioyl- and octadecanedioyl-carnitine, with high dicarboxylycarnitine/monocarboxylylcarnitine ratio. Elevation of very long-chain acylcarnitines C24- and C26-, i.e. lignoceroyl- and cerotoyl-carnitine, was detected in some PBDs and in D-bifunctional protein deficiency. Similar abnormalities were also found in neonatal screening blood spots. Detection of these compounds alone, in the absence of other shorter-chain acylcarnitines, is highly specific and characteristic of PBD, as confirmed by the differing profiles observed in patients with adrenoleukodystrophy and mitochondrial long-chain fatty acid oxidation defects. Our study adds a novel method to the diagnosis of PBD, which may also be of benefit for future neonatal mass screening programs based on acylcarnitine profiling.

Impaired activity of the γ-glutamyl cycle in nephropathic cystinosis fibroblasts

Cystinotic patients have been shown to excrete in their urine high levels of pyroglutamate, an intermediate metabolite of the adenosine triphosphate (ATP)–dependent γ-glutamyl cycle, which is responsible for glutathione (GSH) synthesis. Human fibroblasts were used to study the mechanisms leading to pyroglutamate accumulation in nephropathic cystinosis (NC). We show that inhibition of ATP synthesis caused a marked intracellular accumulation of pyroglutamate, reflecting decreased GSH synthesis. Despite similar degrees of ATP depletion, pyroglutamate increased more in cystinotic fibroblasts than in controls, while GSH decreased to lower levels. In addition, cystinotic cells exposed to oxidative stress (hydrogen peroxide) were unable to increase their GSH concentration above baseline. These results could not be attributed to differences in mitochondrial oxidative activity or to increased apoptotic cell death. Together, these results support the hypothesis that cysteine derived from lysosomal cystine efflux limits the activity of the γ-glutamyl cycle and GSH synthesis.