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Purpose To test the association between death and both qualitative and quantitative CT parameters obtained visually and by software in coronavirus disease (COVID-19) early outbreak. Methods The study analyzed retrospectively patients underwent chest CT at hospital admission for COVID-19 pneumonia suspicion, between February 21 and March 6, 2020. CT was performed in case of hypoxemia or moderate-to-severe dyspnea. CT scans were analyzed for quantitative and qualitative features obtained visually and by software. Cox proportional hazards regression analysis examined the association between variables and overall survival (OS). Three models were built for stratification of mortality risk: clinical, clinical/visual CT evaluation, and clinical/software-based CT assessment. AUC for each model was used to assess performance in predicting death. Results The study included 248 patients (70% males, median age 68 years). Death occurred in 78/248 (32%) patients. Visual pneumonia extent > 40% (HR 2.15, 95% CI 1.2–3.85, P = 0.01), %high attenuation area – 700 HU > 35% (HR 2.17, 95% CI 1.2–3.94, P = 0.01), exudative consolidations (HR 2.85–2.93, 95% CI 1.61–5.05/1.66–5.16, P < 0.001), visual CAC score > 1 (HR 2.76–3.32, 95% CI 1.4–5.45/1.71–6.46, P < 0.01/P < 0.001), and CT classified as COVID-19 and other disease (HR 1.92–2.03, 95% CI 1.01–3.67/1.06–3.9, P = 0.04/P = 0.03) were significantly associated with shorter OS. Models including CT parameters (AUC 0.911–0.913, 95% CI 0.873–0.95/0.875–0.952) were better predictors of death as compared to clinical model (AUC 0.869, 95% CI 0.816–0.922; P = 0.04 for both models). Conclusions In COVID-19 patients, qualitative and quantitative chest CT parameters obtained visually or by software are predictors of mortality. Predictive models including CT metrics were better predictors of death in comparison to clinical model. Supplementary Information The online version contains supplementary material available at 10.1007/s10140-020-01867-1.

Purpose Chest computed tomography (CT) is considered a reliable imaging tool for COVID-19 pneumonia diagnosis, while lung ultrasound (LUS) has emerged as a potential alternative to characterize lung involvement. The aim of the study was to compare diagnostic performance of admission chest CT and LUS for the diagnosis of COVID-19. Methods We included patients admitted to emergency department between February 21-March 6, 2020 (high prevalence group, HP) and between March 30-April 13, 2020 (moderate prevalence group, MP) undergoing LUS and chest CT within 12 h. Chest CT was considered positive in case of “indeterminate”/“typical” pattern for COVID-19 by RSNA classification system. At LUS, thickened pleural line with ≥ three B-lines at least in one zone of the 12 explored was considered positive. Sensitivity, specificity, PPV, NPV, and AUC were calculated for CT and LUS against real-time reverse transcriptase polymerase chain reaction (RT-PCR) and serology as reference standard. Results The study included 486 patients (males 61 %; median age, 70 years): 247 patients in HP (COVID-19 prevalence 94 %) and 239 patients in MP (COVID-19 prevalence 45 %). In HP and MP respectively, sensitivity, specificity, PPV, and NPV were 90–95 %, 43–69 %, 96−72 %, 20–95 % for CT and 94−93 %, 7–31 %, 94−52 %, 7–83 % for LUS. CT demonstrated better performance than LUS in diagnosis of COVID-19, both in HP (AUC 0.75 vs 0.51; P

Organic acidurias are inherited metabolic diseases due to the deficiency of an enzyme or a transport protein involved in one of the several cellular metabolic pathways devoted to the catabolism of amino acids, carbohydrates or lipids. These deficiencies result in abnormal accumulation of organic acids in the body and their abnormal excretion in urine. More than 65 organic acidurias have been described; the incidence varies, individually, from 1 out of 10,000 to >1 out of 1000,000 live births. Collectively, their incidence approximates 1 out of 3000 live births. Among these disorders, methyl malonic aciduria, propionic aciduria, maple syrup urine disease and isovaleric aciduria are sometimes referred to as classical organic acidurias. In this review, we focused on the basic GC–MS-based methodologies employed in the diagnosis of classical organic acidurias and provided updated reference values for the most common involved organic acids. We also attempted to provide the most recent updates on the pathogenetic bases of these diseases.

3-Methylcrotonylglycinuria is an organic aciduria resulting from deficiency of 3-methylcrotonyl-CoA carboxylase (3-MCC), a biotin-dependent mitochondrial enzym carboxylating 3-methylcrotonyl-CoA to 3-methylglutaconyl-CoA during leucine catabolism. Its deficiency, due to mutations on MCCC1 and MCCC2 genes, leads to accumulation of 3-methylcrotonyl-CoA metabolites in blood and/or urine, primarily 3-hydroxyisovaleryl-carnitine (C5-OH) in plasma and 3-methylcrotonyl-glycine (3-MCG) and 3-hydroxyisovaleric acid (3-HIVA) in the urine. The phenotype of 3-MCC deficiency is highly variable, ranging from severe neurological abnormalities and death in infancy to asymptomatic adults. Here we report the biochemical and molecular characterization of an Italian asymptomatic girl, positive for the newborn screening test. Molecular analysis showed two mutations in the MCCC2 gene, an already described missense mutation, c.691A > T (p.I231F), and a novel splicing mutation, c.1150-1G > A. We characterized the expression profile of the splice mutation by functional studies.

Background: Glycogen storage disease type I (GSDI) is an inborn error of carbohydrate metabolism caused by mutations of either the G6PC gene (GSDIa) or the SLC37A4 gene (GSDIb). GSDIa patients are at higher risk of developing insulin-resistance (IR). Mitochondrial dysfunction has been implicated in the development of IR. Mitochondrial dysfunction can demonstrate abnormalities in plama acylcarnitines (ACs) and urine organic acids (UOA). The aim of the study was to investigate the presence of mitochondrial impairment in GSDI patients and its possible connection with IR. Methods: Fourteen GSDIa, seven GSDIb patients, 28 and 14 age and sex-matched controls, were enrolled. Plasma ACs, UOA, and surrogate markers of IR (HOMA-IR, QUICKI, ISI, VAI) were measured. Results: GSDIa patients showed higher short-chain ACs and long-chain ACs levels and increased urinary excretion of lactate, pyruvate, 2-ketoglutarate, 3-methylglutaconate, adipate, suberate, aconitate, ethylmalonate, fumarate, malate, sebacate, 4-octenedioate, 3OH-suberate, and 3-methylglutarate than controls (p < 0.05). GSDIb patients showed higher C0 and C4 levels and increased urinary excretion of lactate, 3-methylglutarate and suberate than controls (p < 0.05). In GSDIa patients C18 levels correlated with insulin serum levels, HOMA-IR, QUICKI, and ISI; long-chain ACs levels correlated with cholesterol, triglycerides, ALT serum levels, and VAI. Discussion: Increased plasma ACs and abnormal UOA profile suggest mitochondrial impairment in GSDIa. Correlation data suggest a possible connection between mitochondrial impairment and IR. We hypothesized that mitochondrial overload might generate by-products potentially affecting the insulin signaling pathway, leading to IR. On the basis of the available data, the possible pathomechanism for IR in GSDIa is proposed.

In the last years tandem mass spectrometry (MS/MS) has become a leading technology used for neonatal screening purposes. Newborn screening by MS/MS on dried blood spot samples (DBS) has one of its items in methionine levels: the knowledge of this parameter allows the identification of infant affected by homocystinuria (cystathionine β-synthase, CBS, deficiency) but can also lead, as side effect, to identify cases of methionine adenosyltransferase (MAT) type I/III deficiency.We started an expanded newborn screening for inborn errors of metabolism in Campania region in 2007. Here we report our ten years experience on expanded newborn screening in identifying patients affected by hypermethioninemia. During this period we screened approximately 77,000 infants and identified two cases: one case of classical homocystinuria and one patient affected by defect of MAT I/III. In this paper we describe these patients and their biochemical follow-up and review the literature concerning worldwide newborn screening reports on incidence of CBS and MAT deficiency. Keywords: Newborn screening, Hypermethioninemia, MAT I/III deficiency, CBS deficiency

Background Aggressive behaviour represents a frequent symptom in people with intellectual disability (PWID). Despite uncertain evidence of effectiveness, the use of antipsychotics (APs) drugs to treat aggressive behaviour is very common. Antipsychotic medication of aggressivity in PWID has recently become one of the most debated issues in mental health and the need of further research is persistently stressed by most researchers. Aim The present study was firstly aimed at evaluating the effectiveness (efficacy on target behaviour, safety and persistence on treatment) of new generation APs, in particular, olanzapine and risperidone in treating aggressive behaviour in PWID for who previous medication with first generation APs (FGAs) were not effective. Methods 62 subjects with intellectual disability underwent to a 2-arm, parallel group pragmatic trial of olanzapine and risperidone with balanced randomisation and blind assessment of outcome at 4, 8, 12, 16, 20 and 24 weeks after a switch (cross-tapering) from a 24-week treatment with FGAs. Aggressive behaviours were assessed by Overt Aggression Scale (OAS) and clinical outcome by Clinical Global Impression Scale. Side effects were assessed with Dosage Record and Treatment Emergent Symptoms Scale, other symptom-specific scales, laboratory and instrumental tests. Results Both risperidone and olanzapine resulted to be more effective than FGAs in reducing aggressive behaviour. Repeated-measures analysis of covariance revealed that treatment groups differed for cumulative number of aggressive episodes during the FGAs treatment, which was higher for olanzapine. Conclusion Our findings seem to confirm that olanzapine and risperidone can be effective in reducing aggressive behaviour in PWID. Both compounds resulted to be well tolerated, with side effects similar to those encountered in other patient populations.

Inborn errors of metabolism are genetic disorders due to impaired activity of enzymes, transporters, or cofactors resulting in accumulation of abnormal metabolites proximal to the metabolic block, lack of essential products or accumulation of by-products. Many of these disorders have serious clinical consequences for affected neonates, and an early diagnosis allows presymptomatic treatment which can prevent severe permanent sequelae and in some cases death. Expanded newborn screening for these diseases is a promising field of targeted metabolomics. Here we report the application, between 2007 and 2014, of this approach to the identification of newborns in southern Italy at risk of developing a potentially fatal disease. The analysis of amino acids and acylcarnitines in dried blood spots by tandem mass spectrometry revealed 24 affected newborns among 45,466 infants evaluated between 48 and 72 hours of life (overall incidence: 1 : 1894). Diagnoses of newborns with elevated metabolites were confirmed by gas chromatography-mass spectrometry, biochemical studies, and genetic analysis. Five infants were diagnosed with medium-chain acyl CoA dehydrogenase deficiency, 1 with methylmalonic acidemia with homocystinuria type CblC, 2 with isolated methylmalonic acidemia, 1 with propionic acidemia, 1 with isovaleric academia, 1 with isobutyryl-CoA dehydrogenase deficiency, 1 with beta ketothiolase deficiency, 1 with short branched chain amino acid deficiency, 1 with 3-methlycrotonyl-CoA carboxylase deficiency, 1 with formimino-transferase cyclodeaminase deficiency, and 1 with cystathionine-beta-synthase deficiency. Seven cases of maternal vitamin B12 deficiency and 1 case of maternal carnitine uptake deficiency were detected. This study supports the widespread application of metabolomic-based newborn screening for these genetic diseases.

The Sanfilippo type A syndrome, one of the most frequent forms of mucopolysaccharidosis III, is characterized by severe mental retardation, progressive neurological degeneration, and mild somatic changes. It is due to a deficiency of heparan-N-sulfatase (sulfamidase) activity and consequent excretion of heparan sulfate in the urine. The disease is transmitted through an autosomal recessive mechanism, and more than 60 gene mutations have been identified. Up to now, only 10 cases of attenuated form of Sanfilippo type A syndrome have been described, and the specific mutation has been identified only in two of them. We report here on a female patient, 20 years old, with Sanfilippo type A syndrome presenting with a mild clinical phenotype characterized essentially by a moderate nonevolving mental retardation. The genetic analysis demonstrated that the patient is homozygous for mutation R206P; presence of polymorphism R456H was also found. This study places R206P as a mild mutation underlying Sanfilippo type A disease.

Mucopolysaccharidosis type IIIB (MPS IIIB; or Sanfilippo syndrome type B) is a lysosomal disease, due to glycosaminoglycan storage caused by mutations on the α-N-acetylglucosaminidase (NAGLU) gene. The disease is characterized by neurological dysfunction but relatively mild somatic manifestations. No effective treatment is available for affected patients. In the present study, we evaluated the role of a lentiviral vector as the transducing agent of NAGLU cDNA in MPS IIIB fibroblasts. The vector expressed high transduction efficiency and high levels of enzymic activity, 20-fold above normal levels, persisting for at least 2 months. PCR experiments confirmed the integration of the viral vector into the target genome. The NAGLU activity restored by virus infection was sufficient to normalize glycosaminoglycan accumulation, which is directly responsible for the disease phenotype. Metabolic labelling experiments on transduced fibroblasts exhibited, in the medium and in cellular lysates, polypeptide forms of 84 and 80kDa respectively related to the precursor and mature forms of the enzyme. The enzyme secreted by transduced MPS IIIB fibroblasts was endocytosed in deficient cells by the mannose 6-phosphate system. Thus we show that lentiviral vectors may provide a therapeutic approach for the treatment of MPS IIIB disease.

Mucopolysaccharidosis type I (MPS I) results from a deficiency in the enzyme alpha-L-iduronidase (IDUA), and is characterized by skeletal abnormalities, hepatosplenomegaly and neurological dysfunction. In this study, we used a late generation lentiviral vector to evaluate the utility of this vector system for the transfer and expression of the human IDUA cDNA in MPS I fibroblasts. We observed that the level of enzyme expression in transduced cells was 1.5-fold the level found in normal cells; the expression persisted for at least two months. In addition, transduced MPS I fibroblasts were capable of clearing intracellular radiolabeled glycosaminoglycan (GAG). Pulse-chase experiments on transduced fibroblasts showed that the recombinant enzyme was synthesized as a 76-kDa precursor form and processed to a 66-kDa mature form; it was released from transduced cells and was endocytosed into a second population of untreated MPS I fibroblasts via a mannose 6-phosphate receptor. These results suggest that the lentiviral vector may be used for the delivery and expression of the IDUA gene to cells in vivo for treatment of MPS I.

Mucopolysaccharidosis type II (Hunter syndrome; OMIM 309900) is a rare X-linked recessive lysosomal storage disorder caused by the deficiency of the enzyme iduronate-2-sulfatase (IDS; EC 3.1.6.13). Different alterations at the IDS locus, mostly missense mutations, have been demonstrated, by expression study, as deleterious, causing significant consequences on the enzyme function or stability. In the present study we report on the results of the transient expression of the novel K347T, 533delTT, N265I and the already described 473delTCC (previously named ΔS117) mutations in the COS 7 cells proving their functional consequence on IDS activity. This type of information is potentially useful for genotype–phenotype correlation, prognosis and possible therapeutic intervention.

Sanfilippo syndrome type B (mucopolysaccharidosis IIIB) is a rare autosomal recessive disorder characterized by the inability to degrade heparan sulfate because of a deficiency of the lysosomal enzyme alpha-N-acetylglucosaminidase (NAGLU). We performed mutation screening in a group of 20 patients, identyifing 28 mutations, 14 of which were novel (L35F, 204delC, 221insGCGCG, G82D, W156C, 507delC, IVS3+1G--A, E336X, V501G, R520W, S534Y, W649C, 1953insGCCA, 2185delAGA). Four of these mutations were found in homozygosity and only one was seen in two different patients, showing the remarkable molecular heterogeneity of the disease. Mutation IVS3+1G--A produces aberrant RNA splicing: it represents a base substitution from G to A of the invariant GT dinucleotides at the splicing donor site of intron 3 resulting in the skipping of exon 3 and both exons 2 and 3. Transient transfection of COS cells, by DNA mutagenized with NAGLU mutations, produced enzymatic molecules without activity, demonstrating the deleterious nature of the defects. Metabolic labeling of transfected mutants suggested a normal synthesis of the involved polypeptide for missense alterations, whereas increased protein or mRNA instability was shown for nonsense and most of the frameshift mutations.

Sanfilippo syndrome type A or mucopolysaccharidosis IIIA (MPS IIIA) results from the deficiency of the enzyme heparan N-sulfatase (NS, EC 3.10.1.1), required for the degradation of heparan sulfate. Molecular defects of 24 Italian MPS IIIA patients were recently reported by our group. We report here two novel mutations: 1040insT and Q365X and the expression studies on 15 of the identified defects. Transient expression of COS cells by cDNA mutagenized to correspond to heparan N-sulfatase mutations Y40N, A44T, 166delG, G122R, P128L, L146P, R150Q, D179N, R182C, R206P, P227R, 1040insT, 1093insG, E369K, R377C did not yield active enzyme, demonstrating the deleterious nature of the mutations. Western blot analysis and metabolic labeling experiments revealed, for cells transfected with wild-type enzyme, a precursor 62-kDa form and a mature 56-kDa form. Western blot resulted, for 11 mutations, in the presence of both forms, indicating a normal maturation of the mutant enzyme. Western blot, metabolic labeling and immunofluorescence experiments suggested, for mutations 166delG, L146P, 1040insT and 1093insG, an increased degradation of the mutant enzymes. (C) 2000 Published by Elsevier Science B.V.

Maroteaux–Lamy syndrome (mucopolysaccharidosis type VI, MPS VI) is an autosomal recessive disorder due to the deficiency of the lysosomal enzyme N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ASB). Mutation analysis in Maroteaux–Lamy syndrome resulted in the identification of approximately 40 molecular defects underlying a great genetic heterogeneity. Here we report five novel mutations in Italian subjects: S65F, P116H, R315Q, Q503X, P531R; each defect was confirmed by restriction enzyme or amplification refractory mutation system (ARMS) analysis. We also performed a three-dimensional (3-D) structure analysis of the alterations identified by us, and of an additional 22 point mutations reported by other groups, in an attempt to draw helpful information about their possible effects on protein conformation.

Mucopolysaccharidosis type I (MPS IH; Hurler syndrome) is a rare genetic disorder that is caused by mutations in the α-L-iduronidase (IDUA) gene, resulting in the deficiency of IDUA enzyme activity and intra-cellular accumulation of glycosaminoglycans. A characteristic skeletal phenotype is one of the many clinical manifestations in Hurler disease. Since the mechanism(s) underlying these skeletal defects are not completely understood, and bone and cartilage are mesenchymal lineages, we focused on the characterization of mesenchymal cells isolated from the bone marrow (BM) of 5 Hurler patients. IDUA-mutated BM stromal cells (BMSC) derived from MPS IH patients exhibited decreased IDUA activity, consistent with the disease genotype. The expansion rate, phenotype, telomerase activity, and differentiation capacity toward adipocytes, osteoblasts, chondrocytes, and smooth muscle cells in vitro of the MPS I BMSC lines were similar to those of BMSC from age-matched normal control donors. MPS I BMSC also had a similar in vivo osteogenic capacity as normal BMSC. However, MPS I BMSC displayed an increased capacity to support osteoclastogenesis, which may correlate with the up-regulation of the RANKL/RANK/OPG molecular pathway in MPS I BMSC compared with normal BMSC.

Mucopolysacccharidosis (MPS) IIIB is an inherited lysosomal storage disorder caused by the deficiency of alpha-N-acetylglucosaminidase (NAGLU). The disease is characterized by mild somatic features and severe neurological involvement with high mortality. Although several therapeutic approaches have been applied to the murine model of the disease, no effective therapy is available for patients. In this study, we used the lentiviral-NAGLU vector to deliver the functional human NAGLU gene into the brain of young adult MPS IIIB mice. We report the restoration of active enzyme with a sustained expression throughout a large portion of the brain, and a significantly improved behavioral performance of treated animals. Moreover, we analyzed the effect of therapy on the expression profile of some genes related to neurotrophic signaling molecules and inflammatory cytokines previously found altered in MPS IIIB mice. At 1 month from treatment, the level of cerebellin 1 (Cbln1) was decreased while the brain-derived neurotrophic factor (Bdnf) expression was increased, both reaching normal values. At 6 months from treatment a significant reduction in the expression of all the inflammation- and oxidative stress-related genes was observed, as well as the maintenance of the correction of the Bdnf gene expression. These results indicate that NAGLU delivery from intracerebral sources has the capacity to alleviate most disease manifestations in MPS IIIB mice; furthermore, Bdnf might be a response-to-therapy biomarker for MPS IIIB.

Sanfilippo syndrome type A or mucopolysaccharidosis IIIA (MPS IIIA) is a lysosomal storage disorder caused by the deficiency of the enzyme heparin sulfamidase (EC 3.10.1.1), required for the degradation of the mucopolysaccharide heparan sulfate. Patients develop central nervous system degeneration resulting in progressive dementia, developmental delay, hyperactivity, and aggressive behaviour; subjects may present a wide spectrum of clinical severity. Here, we report the results from molecular analysis of 24 Italian MPS IIIA patients diagnosed over the last 15 years in our laboratory. Altogether, we were able to characterize 38 out of the 48 (79%) pathogenic alleles. We identified 16 molecular defects, 13 novel. The majority of alterations were missense mutations: on exon two (Y40N; A44T; S66W; R74C), on exon four (G122R; P128L; L146P; R150Q), on exon five (D179N; R182C), on exon six (P227R) and on exon eight (E369K; R377C). Single base pair deletions: on exon two (A52nt-1) and on exon eight (T360nt-1) and one base pair insertion on exon eight (V361nt+1) were also identified. Restriction enzyme or ARMS analyses were used to confirm each alteration. S66W represents the most common alteration in our patients population accounting for 33% of the total alleles. Interestingly, all six patients from Sardinia present this mutation, and five of them are homozygous for this change, suggesting that these subjects may have been derived from a common founder.

Sanfilippo B syndrome (Mucopolysaccharidosis IIIB, MPS IIIB) is a lysosomal storage disease due to mutations in the gene encoding alpha-N-acetylglucosaminidase and is characterized by a severe neurological disorder. Although several studies have been reported for the murine model of the disease, the molecular basis and the sequence of events leading to neurodegeneration remain to be clarified. We previously suggested the possible involvement of the reactive oxygen species in the disease pathogenesis. In the present paper we extended the analysis of oxidative stress by evaluating the production of superoxide ions throughout the CNS and by evaluating the effect of the stress on the cellular macromolecules. These approaches applied to one-month-old, three-month-old and six-month-old mice revealed that oxidative stress is present in the affected cerebrum and cerebellum tissues from one month from birth, and that it results primarily in protein oxidation, both in the cerebrum and cerebellum, with lipid peroxidation, and especially DNA oxidation, appearing milder and restricted essentially to the cerebellum. We also identified additional genes possibly associated with the neuropathology of MPS IIIB disease. Real time RT-PCR analysis revealed an altered expression of the Sod1, Ret, Bmp4, Tgfb, Gzmb and Prf1 genes. Since Gzmb and Prf1 are proteins secreted by NK/cytotoxic T-cells, these data suggest the involvement of cytotoxic cells in the neuronal pathogenesis. Extending our previous study, findings reported in the present paper show that oxidative stress and all the analyzed stress-related pathological changes occur very early in the disease course, most likely before one month of age.

MPS VI (mucopolysaccharidosis type VI) is a lysosomal storage disease in which deficient activity of the enzyme N-acetylgalactosamine 4-sulfatase [ASB (arylsulfatase B)] impairs the stepwise degradation of the GAG (glycosaminoglycan) dermatan sulfate. Clinical studies of ERT (enzyme replacement therapy) by using rhASB (recombinant human ASB) have been reported with promising results. The release of GAG into the urine is currently used as a biomarker of disease, reflecting in some cases disease severity and in all cases therapeutic responsiveness. Using RNA studies in four Italian patients undergoing ERT, we observed that TNFalpha (tumour necrosis factor alpha) might be a biomarker for MPS VI responsive to therapy. In addition to its role as a potential biomarker, TNFalpha expression could provide insights into the possible pathophysiological mechanisms underlying the mucopolysaccharidoses.

Mucopolysaccharidosis IIIB (MPS IIIB; Sanfilippo syndrome type B) is characterized by profound neurological deterioration. Because a murine model of MPS IIIB disease is available, we focused on analysis of gene expression in the brain and cerebellum of 7-month-old MPS IIIB mice by pathway-specific filter microarrays designed to probe apoptotic-related, neurotrophic signalling molecules and inflammatory cytokines and receptors. Moreover, we extended the analysis with real-time PCR performed at 1, 3, 7 months after birth. Bdnf was down-regulated in the brain but up-regulated in the cerebellum at 7 months of age, both at RNA and at protein levels. Cbln1 presented a threefold increase in the oldest brains while remaining unaltered in the cerebellum. Ccl3, Casp11, gp91phox, p67phox, and p47phox showed an increased expression in both brain and cerebellum at each examined time point. Ccl3, in particular, exhibited in both organs and at all times tested approximately a tenfold increase in its expression. Insofar as p47phox, p67phox, and gp91phox are all components of the phagocyte NADPH oxidase, our results suggest the possible involvement of the reactive oxygen species in the genesis of neurodegeneration in MPS IIIB disease. © 2006 Wiley-Liss, Inc.

The Sanfilippo syndrome type B (mucopolysaccharidosis IIIB) is an autosomal recessive disorder due to mutations in the gene encoding NAGLU (α-N-acetylglucosaminidase), one of the enzymes required for the degradation of the GAG (glycosaminoglycan) heparan sulphate. No therapy exists for affected patients. We have shown previously the efficacy of lentiviral-NAGLU-mediated gene transfer in correcting in vitro the defect on fibroblasts of patients. In the present study, we tested the therapy in vivo on a knockout mouse model using intravenous injections. Mice (8–10 weeks old) were injected with one of the lentiviral doses through the tail vein and analysed 1 month after treatment. A single injection of lentiviral-NAGLU vector resulted in transgene expression in liver, spleen, lung and heart of treated mice, with the highest level reached in liver and spleen. Expression of 1% normal NAGLU activity in liver resulted in a 77% decrease in the GAG content; more remarkably, an expression of 0.16% normal activity in lung was capable of decreasing the GAG level by 29%. Long-term (6 months) follow up of the gene therapy revealed that the viral genome integration persisted in the target tissues, although the real-time PCR analysis showed a decrease in the vector DNA content with time. Interestingly, the decrease in GAG levels was maintained in liver, spleen, lung and heart of treated mice. These results show the promising potential and the limitations of lentiviral-NAGLU vector to deliver the human NAGLU gene in vivo.

Mucopolysaccharidosis type I is a lysosomal disease due to mutations in the IDUA gene, resulting in deficiency of alpha- L -iduronidase and accumulation of glycosaminoglycans (GAGs). Bone marrow transplantation and enzyme replacement are two therapies considered only moderately successful for affected patients, making the development of novel treatments necessary. We have previously shown the efficacy of lentivirus-mediated gene transfer to correct patient fibroblasts in vitro. Here we tested lentiviral-IDUA vector gene therapy in vivo on a murine MPS I model. Eight- to 10 week-old mice were injected with increasing lentiviral doses via the tail vein and analyzed 1 month after treatment. A single injection of lentiviral-IDUA vector resulted in transgene expression in several murine tissues, with the highest level reached in liver and spleen. Expression of 1% normal activity was sufficient in treated animals to normalize the GAG level in urine, liver, and spleen and was able to reduce the GAG level in kidney, heart, and lung. Polymerase chain reaction assays showed integration of the viral genome only in liver and spleen of treated animals, suggesting that the correction of the pathology in other tissues was due to secretion into the plasma by liver and spleen and uptake of corrective enzyme by distant tissues. Long-term (6 months) analysis showed the presence of enzyme-specific antibodies and the loss of enzyme activity and vector sequence in the target tissue, suggesting that the transgene-specific immune response interfered with long-term therapeutic correction and led to clearance of transduced cells. In conclusion, our results show the promising potential and the limitations of lentiviral-IDUA vector-mediated gene therapy in an in vivo model.

Hypochondroplasia and achondroplasia are skeletal dysplasias, characterized by autosomal dominant inheritance and disproportionate short stature, which occurs mainly due to growth failure of the extremities. Both dysplasias have been mapped to fibroblast growth factor receptor 3 (FGFR3) gene. For hypochondroplasia, two point mutations, both responsible for the Asn540Lys substitution in the region coding the tyrosine kinase domain have been reported. Here we report an A to G transition at position 1651, predicting an Ile538Val substitution in the FGFR3, in hypochondroplasia. The substitution is found in a swedish family with three affected members. The criteria for hypochondroplasia were disproportionate short stature and radiological evidence of shortened long bones and decrease or absence of normal increase in interpedicular distances of the lumbar column. The mutation was detected by direct sequencing and restriction enzyme Tai I digestion. The base change was not found in the FGFR3 genes of unaffected members of the family nor in seventy-five unrelated unaffected individuals, suggesting that it was not a polymorphism. The Ile538Val substitution is a conservative amino acid change (a hydrophobic amino acid incorporated for another hydrophobic amino acid). Nevertheless, it is located in the stretch of nine amino acids, which is highly conserved among all the human fibroblast growth factor receptors. Considering the location of this substitution and the segregation with the phenotype in this family, we propose that it is a causative mutation of hypochondroplasia. It is difficult to establish whether the Ile538Val substitution is rare in hypochondroplasia patients or whether the individuals, who have a moderate degree of short stature, rarely seek medical help for the short stature and consequently are rarely diagnosed as affected by hypochondroplasia.

Nosographic, clinical, therapeutic and psychosocial aspects of psychopathological disorders in 43 mentally retarded patients followed from 1988 to 1991 are presented. Mental problems in mentally retarded subjects are linked to mental illness but do not form a continuum with it. The prognosis is generally favourable. Great attention must be given to the psychosocial aspects as social integration is almost always at risk in the mentally retarded.

SC-41930, 7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxy]-3,4- dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid, is a selective, orally active, LTB4 receptor antagonist currently in clinical trials for psoriasis and ulcerative colitis. Exhaustive SAR studies found a potential backup compound, SC-50605, which was 7-16 times more potent than SC-41930 in LTB4 receptor binding, chemotaxis and degranulation assays. SC-50605 also inhibited LTB4-induced intradermal chemotaxis in cavine skin at an oral dose of 0.10 mg/kg and displayed good activity in animal models of colitis and epidermal inflammation both orally and topically.

Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is a congenital storage disorder resulting from mutations on the iduronate-2-sulfatase (IDS) gene. The disease shows variable clinical phenotypes from severe to mild with progressive neurological dysfunction. The therapeutic options for treatment of MPS II are limited and currently no specific therapies are available; the problem is further compounded by difficulties in delivering therapeutic agents to the central nervous system (CNS). In this work, as a potential treatment for this disease, the transfer of the recombinant IDS enzyme into brain cells has been studied in vitro. Two different approaches to obtain recombinant IDS have been utilized: production of the recombinant enzyme by a transfected human clone (Bosc 23 cells); production of the recombinant enzyme by adenoviral transduction of neuronal (SK-N-BE) or glial (C6) cells. Our data indicate that the transfected as well as the infected cells produce a large amount of the IDS enzyme, which is efficiently endocytosed into neuronal and glial cells through the mannose 6-phosphate (M6P) receptor system. Somatic gene therapy appears therefore to be suitable to correct IDS deficiency in brain cells.

Anal fissure is one of the most common and painful proctologic diseases. Its treatment has long been discussed and several different therapeutic options have been proposed. In the last decades, the understanding of its pathophysiology has led to a progressive reduction of invasive and potentially invalidating treatments in favor of conservative treatment based on anal sphincter muscle relaxation. Despite some systematic reviews and an American position statement, there is ongoing debate about the best treatment for anal fissure. This review is aimed at identifying the best treatment option drawing on evidence-based medicine and on the expert advice of 6 colorectal surgeons with extensive experience in this field in order to produce an Italian position statement for anal fissures. While there is little chance of a cure with conservative behavioral therapy, medical treatment with calcium channel blockers, diltiazem and nifepidine or glyceryl trinitrate, had a considerable success rate ranging from 50 to 90%. Use of 0.4% glyceryl trinitrate in standardized fashion seems to have the best results despite a higher percentage of headache, while the use of botulinum toxin had inconsistent results. Nonresponding patients should undergo lateral internal sphincterotomy. The risk of incontinence after this procedure seems to have been overemphasized in the past. Only a carefully selected group of patients, without anal hypertonia, could benefit from anoplasty.

Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is a lysosomal disease caused by the deficiency of the enzyme iduronate-2-sulfatase (IDS, EC 3.1.6.13). Affected patients show a wide spectrum of clinical phenotypes, from severe to mild. Mutational analysis on this disease resulted in the identification of more than 200 alterations. Bone marrow transplantation (BMT) is considered, at present, an appropriate therapy for MPS II subjects without severe neuropsychological impairment, however molecular analysis in BMT treated patients has been poorly studied. We describe here a patient subjected to BMT in 1995 whose IDS gene alteration, mutation P266H, was identified thereafter. The 4-year follow-up included clinical, biochemical and molecular parameters. DNA analysis showed, after BMT, coexisting host mutant and donor normal alleles, ensuring the effectiveness of the therapy and providing a fast and accurate tool to monitor the colonization of donor cells after treatment.