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1.
J Inherit Metab Dis ; 42(1): 128-139, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30740731

RESUMO

PURPOSE: To assess how the current practice of newborn screening (NBS) for homocystinurias compares with published recommendations. METHODS: Twenty-two of 32 NBS programmes from 18 countries screened for at least one form of homocystinuria. Centres provided pseudonymised NBS data from patients with cystathionine beta-synthase deficiency (CBSD, n = 19), methionine adenosyltransferase I/III deficiency (MATI/IIID, n = 28), combined remethylation disorder (cRMD, n = 56) and isolated remethylation disorder (iRMD), including methylenetetrahydrofolate reductase deficiency (MTHFRD) (n = 8). Markers and decision limits were converted to multiples of the median (MoM) to allow comparison between centres. RESULTS: NBS programmes, algorithms and decision limits varied considerably. Only nine centres used the recommended second-tier marker total homocysteine (tHcy). The median decision limits of all centres were ≥ 2.35 for high and ≤ 0.44 MoM for low methionine, ≥ 1.95 for high and ≤ 0.47 MoM for low methionine/phenylalanine, ≥ 2.54 for high propionylcarnitine and ≥ 2.78 MoM for propionylcarnitine/acetylcarnitine. These decision limits alone had a 100%, 100%, 86% and 84% sensitivity for the detection of CBSD, MATI/IIID, iRMD and cRMD, respectively, but failed to detect six individuals with cRMD. To enhance sensitivity and decrease second-tier testing costs, we further adapted these decision limits using the data of 15 000 healthy newborns. CONCLUSIONS: Due to the favorable outcome of early treated patients, NBS for homocystinurias is recommended. To improve NBS, decision limits should be revised considering the population median. Relevant markers should be combined; use of the postanalytical tools offered by the CLIR project (Collaborative Laboratory Integrated Reports, which considers, for example, birth weight and gestational age) is recommended. tHcy and methylmalonic acid should be implemented as second-tier markers.

2.
Front Neurol ; 9: 605, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30254599

RESUMO

Neuroinflammation has a key role in the pathogenesis of perinatal brain injury. Caffeine, a nonspecific antagonist of adenosine receptors (ARs), is widely used to treat apnea of prematurity and has been linked to a decrease in the incidence of cerebral palsy in premature infants. The mechanisms explaining its neuroprotective effect have not yet been elucidated. The objective of this study was to characterize the expression of adenosine and ARs in two neonatal rat models of neuroinflammation and to determine the effect of A2aR blockade on microglial activation assessed through inflammatory cytokine gene expression. We have used two rat models of microglial activation: the gestational low protein diet (LPD) model, associated with chronic brain injury, and postnatal ibotenate intracerebral injections, responsible for acute excitotoxicity injury. Adenosine blood levels have been measured by Tandem Mass Spectrometry. The expression of ARs in vivo was assessed using qPCR and immunohistochemistry. In vivo models have been replicated in vitro on primary microglial cell cultures exposed to A2aR agonist CGS-21680 or antagonist SCH-58261. The effects of these treatments have been assessed on the M1/M2 cytokine expressions measured by RT-qPCR. LPD during pregnancy was associated with higher adenosine levels in pups at postnatal day 1 and 4. A2aR mRNA expression was significantly increased in both cortex and magnetically sorted microglial cells from LPD animals compared to controls. CD73 expression, responsible for extracellular production of brain adenosine, was significantly increased in LPD cortex and sorted microglia cells. Moreover, CD73 protein level was increased in ibotenate treated animals. In vitro experiments confirmed that LPD or control microglial cells exposed to ibotenate display an increased expression, at both protein and molecular levels, of A2aR and M1 markers (IL-1ß, IL-6, iNOS, TNFα). This pro-inflammatory profile was significantly reduced by SCH-58261, which reduces M1 markers in both LPD and ibotenate-exposed cells, with no effect on control cells. In the same experimental conditions, a partial increased of M1 cytokines was observed in response to A2aR agonist CGS-21680. These results support the involvement of adenosine and particularly of its receptor A2aR in the regulation of microglia in two different animal models of neuroinflammation.

3.
Med Hypotheses ; 90: 53-6, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27063086

RESUMO

Our preliminary data show high levels of adenosine in the blood of very low birth weight (VLBW) infants, positively correlating to their prematurity (i.e. body weight class). This prompted us to look for a mechanism promoting such impressive adenosine increase. We hypothesized a correlation with oxygen challenge. In fact, it is recognized that either oxygen lack or its excess contribute to the pathogenesis of the injuries of prematurity, such as retinopathy (ROP) and periventricular white matter lesions (PWMI). The optimal concentration of oxygen for resuscitation of VLBW infants is currently under revision. We propose that the elevated adenosine blood concentrations of VLBW infants recognizes two sources. The first could be its activity-dependent release from unmyelinated brain axons. Adenosine in this respect would be an end-product of the hypometabolic VLBW newborn unmyelinated axon intensely firing in response to the environmental stimuli consequent to premature birth. Adenosine would be eventually found in the blood due to blood-brain barrier immaturity. In fact, adenosine is the primary activity-dependent signal promoting differentiation of premyelinating oligodendrocyte progenitor cells (OPC) into myelinating cells in the Central Nervous System, while inhibiting their proliferation and inhibiting synaptic function. The second, would be the ecto-cellular ATP synthesized by the endothelial cell plasmalemma exposed to ambient oxygen concentrations due to premature breathing, especially in lung. ATP would be rapidly transformed into adenosine by the ectonucleotidase activities such as NTPDase I (CD39), and NT5E (CD73). An ectopic extra-mitochondrial aerobic ATP synthetic ability was reported in many cell plasma-membranes, among which endothelial cells. The potential implications of the cited hypotheses for the neonatology area would be great. The amount of oxygen administration for reviving of newborns would find a molecular basis for its assessment. VLBW infants may be regarded as those in which premature exposure to ambient oxygen concentrations and oxidative stress causes a premature functioning of the extra-mitochondrial oxidative phosphorylation primarily in axons and endothelium. Adenosine may become a biomarker of prematurity risk, whose implications further studies may assess.


Assuntos
Adenosina/sangue , Recém-Nascido Prematuro/sangue , Recém-Nascido de muito Baixo Peso/sangue , Modelos Biológicos , Potenciais de Ação , Trifosfato de Adenosina/biossíntese , Axônios/metabolismo , Barreira Hematoencefálica , Membrana Celular/metabolismo , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Humanos , Recém-Nascido , Bainha de Mielina/fisiologia , Neurogênese , Oligodendroglia/citologia , Fosforilação Oxidativa , Estresse Oxidativo , Oxigênio/administração & dosagem , Oxigênio/efeitos adversos , Oxigênio/sangue , Oxigenoterapia/efeitos adversos
4.
Carcinogenesis ; 36(11): 1354-62, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26363032

RESUMO

Arginase (ARG) is a metabolic enzyme present in two isoforms that hydrolyze l-arginine to urea and ornithine. In humans, ARG isoform 1 is also expressed in cells of the myeloid lineage. ARG activity promotes tumour growth and inhibits T lymphocyte activation. However, the two ARG transgenic mouse lines produced so far failed to show such effects. We have generated, in two different genetic backgrounds, transgenic mice constitutively expressing ARG1 under the control of the CD68 promoter in macrophages and monocytes. Both heterozygous and homozygous transgenic mice showed a relevant increase in mortality at early age, compared with wild-type siblings (67/267 and 48/181 versus 8/149, respectively, both P < 0.005). This increase was due to high incidence of haematologic malignancies, in particular myeloid leukaemia, myeloid dysplasia, lymphomas and disseminated intravascular coagulation (DIC), diseases that were absent in wild-type mice. Atrophy of lymphoid organs due to reduction in T-cell compartment was also detected. Our results indicate that ARG activity may participate in the pathogenesis of lymphoproliferative and myeloproliferative disorders, suggest the involvement of alterations of L-arginine metabolism in the onset of DIC and confirm a role for the enzyme in regulating T-cell homeostasis.


Assuntos
Arginase/metabolismo , Transtornos Linfoproliferativos/enzimologia , Monócitos/enzimologia , Transtornos Mieloproliferativos/enzimologia , Animais , Arginase/genética , Linhagem da Célula , Feminino , Expressão Gênica , Ativação Linfocitária , Transtornos Linfoproliferativos/patologia , Masculino , Camundongos Transgênicos , Transtornos Mieloproliferativos/patologia , Linfócitos T/enzimologia , Linfócitos T/imunologia
5.
Clin Biochem ; 47(13-14): 1293-6, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24945105

RESUMO

OBJECTIVES: Gaucher disease (GD) diagnosis relies on the demonstration of deficient ß-D-glucosidase (GBA) activity in cellular homogenates. Diagnosis process, however, can be delayed as (i) some GD symptoms are non-specific; and (ii) diagnostic tests are performed in specialized laboratories. These difficulties negatively impact on timely access of patients to therapy. GBA assay in dried blood spots (DBS) represents a method facilitating early identification of patients who will be finally diagnosed with gold standard assay of nucleated cells. Aim of this study is to investigate the DBS analytical performance compared with gold standard method. DESIGN & METHODS: A cross-sectional study started by comparing data of 50 DBS and 50 homogenate samples from the same subjects (25 known-GD and 25 controls). The subsequent phase examined 443 DBS samples. Along with these, 73 blood samples were sent for leukocyte separation and/or EBV-lymphoblast cell lines, and 1 skin biopsy for fibroblast cell lines. Overall the study included a total of 493 subjects. RESULTS: While the results from this first validation group did not yield false positive/negative values, when the analysis was extended to 443 DBS, 14.4% (64 samples) of positive results was yielded. Among these, only 15 were confirmed as GD values with gold standard test. In addition, a thorough examination of some clinical data also revealed 2 false negative results which were confirmed by both enzymatic and molecular analyses. CONCLUSIONS: DBS test could be useful as screening method although with cautions, whereas the standardized GBA assay should remain the gold standard for laboratory diagnosis of Gaucher disease.


Assuntos
Doença de Gaucher/sangue , beta-Glucosidase/sangue , Adolescente , Adulto , Bioensaio/métodos , Coleta de Amostras Sanguíneas/métodos , Linhagem Celular , Criança , Estudos Transversais , Teste em Amostras de Sangue Seco/métodos , Feminino , Humanos , Masculino , Adulto Jovem
6.
Ital J Pediatr ; 39: 33, 2013 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-23705938

RESUMO

3-hydroxy-3-methylglutaric aciduria (OMIM 246450) is a rare autosomal recessive inborn of metabolism due to the deficiency of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase, an enzyme involved both in the ketogenic pathway and leucine catabolism. Acute decompensations present with lethargy, cianosis, hypotonia, vomiting and metabolic acidosis with hypoketotic hypoglycemia. We report the case of a 3 days male with sudden hypoglycemic crisis initially misdiagnosed as a sepsis. HMG-CoA lyase deficiency was achieved through acyl-carnitines profile (showing a typical increasing of 3-hydroxy-isovaleryl and 3-methylgluraryl carnitines) and urinary organic acids analysis (disclosing elevation of 3-hydroxy-3-methylglutaric, 3-methyl-glutaconic, 3-methylglutaric and 3-hydroxyisovaleric acids). This case underlines the need of suspecting such inborn metabolic disorder in cases with hypoglycemia and metabolic acidosis. Acyl-carnitine and urinary organic acids profiles are essential to achieve a prompt diagnosis of treatable metabolic disorders in order to prevent their acute crisis with serious or even fatal consequences.


Assuntos
Acetil-CoA C-Acetiltransferase/deficiência , Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico , Acidose/etiologia , Doença Aguda , Glutaratos/urina , Glicina/análogos & derivados , Glicina/urina , Humanos , Hipoglicemia/etiologia , Recém-Nascido , Masculino , Meglutol/análogos & derivados , Meglutol/urina
7.
Ital J Pediatr ; 38: 59, 2012 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-23095120

RESUMO

Medium chain acyl CoA dehydrogenase deficiency (MCAD) is the most common inborn error of fatty acid oxidation. This condition may lead to cellular energy shortage and cause severe clinical events such as hypoketotic hypoglycemia, Reye syndrome and sudden death. MCAD deficiency usually presents around three to six months of life, following catabolic stress as intercurrent infections or prolonged fasting, whilst neonatal-onset of the disease is quite rare. We report the case of an apparently healthy newborn who suddenly died at the third day of life, in which the diagnosis of MCAD deficiency was possible through peri-mortem blood-spot acylcarnitine analysis that showed very high concentrations of octanoylcarnitine. Genetic analysis at the ACADM locus confirmed the biochemical findings by demonstrating the presence in homozygosity of the frame-shift c.244dup1 (p.Trp82LeufsX23) mutation, a severe genotype that may explain the unusual and very early fatal outcome in this newborn. This report confirms that inborn errors of fatty acid oxidation represent one of the genetic causes of sudden unexpected deaths in infancy (SUDI) and underlines the importance to include systematically specific metabolic screening in any neonatal unexpected death.


Assuntos
Erros Inatos do Metabolismo Lipídico/genética , Morte Súbita do Lactente , Acil-CoA Desidrogenase/deficiência , Acil-CoA Desidrogenase/genética , Consanguinidade , Evolução Fatal , Genótipo , Humanos , Recém-Nascido
8.
Am J Pathol ; 178(3): 1257-69, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21356376

RESUMO

The metabotropic glutamate (mGlu) receptor 1 (GRM1) has been shown to play an important role in neuronal cells by triggering, through calcium release from intracellular stores, various signaling pathways that finally modulate neuron excitability, synaptic plasticity, and mechanisms of feedback regulation of neurotransmitter release. Herein, we show that Grm1 is expressed in glomerular podocytes and that a glomerular phenotype is exhibited by Grm1(crv4) mice carrying a spontaneous recessive inactivating mutation of the gene. Homozygous Grm1(crv4/crv4) and, to a lesser extent, heterozygous mice show albuminuria, podocyte foot process effacement, and reduced levels of nephrin and other proteins known to contribute to the maintenance of podocyte cell structure. Overall, the present data extend the role of mGlu1 receptor to the glomerular filtration barrier. The regulatory action of mGlu1 receptor in dendritic spine morphology and in the control of glutamate release is well acknowledged in neuronal cells. Analogously, we speculate that mGlu1 receptor may regulate foot process morphology and intercellular signaling in the podocyte.


Assuntos
Albuminúria/patologia , Glomérulos Renais/patologia , Receptores de Glutamato Metabotrópico/deficiência , Albuminúria/complicações , Albuminúria/metabolismo , Animais , Western Blotting , Células Cultivadas , Doxorrubicina/farmacologia , Imunofluorescência , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Córtex Renal/efeitos dos fármacos , Córtex Renal/metabolismo , Córtex Renal/patologia , Nefropatias/complicações , Nefropatias/metabolismo , Nefropatias/patologia , Glomérulos Renais/metabolismo , Glomérulos Renais/ultraestrutura , Camundongos , Fenótipo , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Podócitos/patologia , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
9.
Genet Med ; 13(3): 230-54, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21325949

RESUMO

PURPOSE: To achieve clinical validation of cutoff values for newborn screening by tandem mass spectrometry through a worldwide collaborative effort. METHODS: Cumulative percentiles of amino acids and acylcarnitines in dried blood spots of approximately 25­30 million normal newborns and 10,742 deidentified true positive cases are compared to assign clinical significance, which is achieved when the median of a disorder range is, and usually markedly outside, either the 99th or the 1st percentile of the normal population. The cutoff target ranges of analytes and ratios are then defined as the interval between selected percentiles of the two populations. When overlaps occur, adjustments are made to maximize sensitivity and specificity taking all available factors into consideration. RESULTS: As of December 1, 2010, 130 sites in 45 countries have uploaded a total of 25,114 percentile data points, 565,232 analyte results of true positive cases with 64 conditions, and 5,341 cutoff values. The average rate of submission of true positive cases between December 1, 2008, and December 1, 2010, was 5.1 cases/day. This cumulative evidence generated 91 high and 23 low cutoff target ranges. The overall proportion of cutoff values within the respective target range was 42% (2,269/5,341). CONCLUSION: An unprecedented level of cooperation and collaboration has allowed the objective definition of cutoff target ranges for 114 markers to be applied to newborn screening of rare metabolic disorders.


Assuntos
Doenças Metabólicas/diagnóstico , Triagem Neonatal , Espectrometria de Massas em Tandem , Aminoácidos/sangue , Carnitina/análogos & derivados , Carnitina/sangue , Humanos , Recém-Nascido , Cooperação Internacional , Valores de Referência , Sensibilidade e Especificidade , Software
10.
Am J Med Genet A ; 146A(13): 1676-81, 2008 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-18536048

RESUMO

We report on two new patients with straight-chain acyl-coenzyme A oxidase deficiency. Early onset hypotonia, seizures and psychomotor delay were observed in both cases. Plasma very-long-chain fatty acids were abnormal in both patients, whereas the plasma levels of phytanic acid, pristanic acid, the bile acid intermediates DHCA and THCA, and erythrocyte plasmalogen levels were normal. Studies in fibroblasts from the two patients revealed a deficiency of one of the two peroxisomal acyl-CoA oxidases, that is, straight-chain acyl-CoA oxidase (ACOX1). Subsequent molecular analysis of ACOX1 showed a homozygous deletion, which removes a large part of intron 3 and exons 4-14 in the first patient. Mutation analysis in the second patient revealed compound heterozygosity for two mutations, including: (1) a c.692 G > T (p.G231V) mutation and (2) skipping of exon 13 (c.1729_1935del (p.G577_E645del).


Assuntos
Acil-CoA Oxidase/deficiência , Acil-CoA Oxidase/genética , Transtornos Peroxissômicos/enzimologia , Transtornos Peroxissômicos/genética , Sequência de Bases , Encéfalo/anormalidades , Pré-Escolar , Consanguinidade , DNA/genética , Feminino , Genes Recessivos , Homozigoto , Humanos , Recém-Nascido , Imagem por Ressonância Magnética , Transtornos Peroxissômicos/diagnóstico , Peroxissomos/enzimologia , Gravidez , Diagnóstico Pré-Natal , Deleção de Sequência
11.
Int J Cancer ; 123(5): 1108-16, 2008 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-18528866

RESUMO

In human prostate cancer, Arginase 2 (ARG2) and nitric oxide synthase (NOS) are concomitantly expressed by tumor cells, and induce tumor immune escape via peroxynitrite-dependent Tyrosine nitrosylation. Since there were no data regarding this immune suppressive mechanism in other tumor types, and an evaluation of its clinical relevance in human tumors had still to be provided, we have investigated presence and clinical relevance of ARG2 and NOS expression in lung cancer. No evidence of NOS expression was found, no significant NOS enzymatic activity was detected. Instead, ARG2 protein was expressed by tumor cells. In a cohort of 120 patients, the amount of ARG2-positive tumor cells was significantly higher in small cell lung cancers (SCLC) than in non-small cell lung cancers (NSCLC). Large cell undifferentiated carcinomas had twice ARG2 than the other NSCLC subtypes. ARG2 expression was increased in Grade 3 tumors, as compared to Grades 1 and 2. However, no relationship was found with tumor size and stage, and with patient survival. Indeed, the enzyme was active, since the Arginine catabolite Ornithine was produced, but Arginine depletion was not attained. In addition, nitrotyrosine was not found in tumor tissue. Accordingly, when tumor cells isolated from lung cancer were incubated with activated autologous T cells, no inhibition of proliferation was detected. Our results indicate that ARG2 is expressed in lung cancer, but it does not induce tumor immune escape and does not affect disease progression, most probably due to the lack of concomitant NOS expression.


Assuntos
Arginase/metabolismo , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma de Células Pequenas/enzimologia , Carcinoma de Células Pequenas/imunologia , Proliferação de Células , Progressão da Doença , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Itália , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Óxido Nítrico Sintase/metabolismo , Nitritos/metabolismo , Ácido Peroxinitroso/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T , Tirosina/análogos & derivados , Tirosina/biossíntese
12.
Pediatr Nephrol ; 22(5): 727-33, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17277954

RESUMO

We prospectively evaluated the effects of L-carnitine supplementation on plasma free carnitine (FC) levels, serum lipid profile, and erythropoietin (rhEPO) requirement in 24 children treated with peritoneal dialysis (PD; n=16) or hemodialysis (HD; n=8). The study was divided into a 3-month observation period, and a 3-month treatment period during which patients received 20 mg/kg per day of L-carnitine given orally. Clinical, biochemical, and hematological data were collected every 3 months. FC levels were measured in plasma and peritoneal dialysate by tandem mass spectrometry. There were no statistically significant changes in lipid levels, hemoglobin, or rhEPO requirements during the course of the study. Fifteen patients (13 PD, 2 HD) had plasma FC levels measured before and after treatment; FC levels increased from 32.1 +/- 14.1 micromol/l to 80.9 +/- 38.7 micromol/l (P<0.001). In PD patients, dialysate FC losses increased from 106 +/- 78 micromol/day at baseline to 178 +/- 119 micromol/day after supplementation. Positive correlations between FC plasma levels and dialysate levels (R=0.507) or daily excretion (R=0.603) were found after treatment. In our case series, an oral dose of 20 mg/kg per day of L-carnitine restored FC levels and produced a positive carnitine balance with no significant effects on hematological parameters or lipid profile over a 3-month period. Prolonged treatment duration may be required to obtain significant results.


Assuntos
Anemia/sangue , Carnitina/uso terapêutico , Lipídeos/sangue , Diálise Peritoneal , Diálise Renal , Adolescente , Anemia/tratamento farmacológico , Carnitina/administração & dosagem , Carnitina/sangue , Criança , Suplementos Nutricionais , Eritropoetina/uso terapêutico , Hematócrito , Humanos , Seleção de Pacientes , Estudos Prospectivos
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