RESUMEN
Diagnosis and cure for rare diseases represent a great challenge for the scientific community who often comes up against the complexity and heterogeneity of clinical picture associated to a high cost and time-consuming drug development processes. Here we show a drug repurposing strategy applied to nephropathic cystinosis, a rare inherited disorder belonging to the lysosomal storage diseases. This approach consists in combining mechanism-based and cell-based screenings, coupled with an affordable computational analysis, which could result very useful to predict therapeutic responses at both molecular and system levels. Then, we identified potential drugs and metabolic pathways relevant for the pathophysiology of nephropathic cystinosis by comparing gene-expression signature of drugs that share common mechanisms of action or that involve similar pathways with the disease gene-expression signature achieved with RNA-seq.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/genética , Cistinosis/tratamiento farmacológico , Cistinosis/genética , Reposicionamiento de Medicamentos , Enfermedades Renales/tratamiento farmacológico , Enfermedades Renales/genética , Enfermedades Raras/tratamiento farmacológico , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/efectos de la radiación , Células Cultivadas , Biología Computacional/métodos , Cistinosis/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Humanos , Enfermedades Renales/metabolismo , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Redes y Vías Metabólicas , Enfermedades Raras/genética , Enfermedades Raras/metabolismo , TranscriptomaRESUMEN
Cystinosis is a rare lysosomal storage disorder caused by loss-of-function mutations of the CTNS gene, encoding cystinosin, a symporter that mediates cystine efflux from lysosomes. Approximately 95% of patients with cystinosis display renal Fanconi syndrome, short stature, osteopenia, and rickets. In this study, we investigated whether the absence of cystinosin primarily affects bone remodeling activity, apart from the influences of the Fanconi syndrome on bone mineral metabolism. Using micro-computed tomography and histomorphometric and bone serum biomarker analysis, we evaluated the bone phenotype of 1-month-old Ctns-/- knockout (KO) male mice without tubulopathy. An in vitro study was performed to characterize the effects of cystinosin deficiency on osteoblasts and osteoclasts. Micro-computed tomography analysis showed a reduction of trabecular bone volume, bone mineral density, and number and thickness in KO mice compared with wild-type animals; histomorphometric analysis revealed a reduction of osteoblast and osteoclast parameters in tibiae of cystinotic mice. Decreased levels of serum procollagen type 1 amino-terminal propeptide and tartrate-resistant acid phosphatase in KO mice confirmed reduced bone remodeling activity. In vitro experiments showed an impairment of Ctns-/- osteoblasts and osteoclasts. In conclusion, cystinosin deficiency primarily affects bone cells, leading to a bone loss phenotype of KO mice, independent from renal failure.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/fisiología , Enfermedades Óseas/patología , Cistinosis/patología , Osteoblastos/patología , Osteogénesis , Animales , Enfermedades Óseas/etiología , Enfermedades Óseas/metabolismo , Cistinosis/etiología , Cistinosis/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoblastos/metabolismoRESUMEN
Methylmalonic acidemia with homocystinuria, cobalamin deficiency type C (cblC) (MMACHC) is the most common inborn error of cobalamin metabolism. Despite a multidrug treatment, the long-term follow-up of early-onset patients is often unsatisfactory, with progression of neurological and ocular impairment. Here, the in-vivo proteome of control and MMACHC lymphocytes (obtained from patients under standard treatment with OHCbl, betaine, folate and L-carnitine) was quantitatively examined by two dimensional differential in-gel electrophoresis (2D-DIGE) and mass spectrometry. Twenty three proteins were found up-regulated and 38 proteins were down-regulated. Consistent with in vivo studies showing disturbance of glutathione metabolism, a deregulation in proteins involved in cellular detoxification, especially in glutathione metabolism was found. In addition, relevant changes were observed in the expression levels of proteins involved in intracellular trafficking and protein folding, energy metabolism, cytoskeleton organization and assembly. This study demonstrates relevant changes in the proteome profile of circulating lymphocytes isolated from treated cblC patients. Some results confirm previous observations in vivo on fibroblast, thus concluding that some dysregulation is ubiquitous. On the other hand, new findings could be tissue-specific. These observations expand our current understanding of the cblC disease and may ignite new research and therapeutic strategies to treat this disorder.
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Homocistinuria/metabolismo , Proteoma/análisis , Deficiencia de Vitamina B 12/congénito , Adolescente , Estudios de Casos y Controles , Niño , Preescolar , Electroforesis en Gel Bidimensional , Femenino , Homocistinuria/sangre , Humanos , Lactante , Linfocitos/metabolismo , Linfocitos/patología , Masculino , Proteómica , Transducción de Señal , Deficiencia de Vitamina B 12/sangre , Deficiencia de Vitamina B 12/metabolismoRESUMEN
BACKGROUND: Treatment with folic acid and vitamin B12 appears capable of reducing total plasma homocysteine levels (tHcy), but it is unknown whether vitamin B12 alone reduces tHcy values. In this study we investigate the effects of alternate vitamins supplementation on homocysteine levels in patients treated by diffusive and convective dialysis techniques. METHODS: 74 patients were randomized blindly into two groups of 37 subjects each. The first group was treated initially with vitamin B12 for 2 months and with folic acid for the following 2 months. The second group was treated initially with folic acid. A wash out period of 2 months followed the treatment in both groups. RESULTS: Total homocysteine levels decreased in both groups following the alternate vitamins therapy and dialysis, without significant difference between diffusive and convective techniques. Surprisingly, after the wash-out period, tHcy increased remarkably, regardless of the dialysis procedure used. At the end of the study, folate levels showed a higher reduction with haemodialysis compared to haemodiafiltration. In contrast, vitamin B12 levels showed a significant increase using diffusive haemodialysis, confirming a decisive role of membrane performance. CONCLUSIONS: In conclusion we show for the first time that, even if total homocysteine levels decreased in both dialysis procedures, the convective techniques demonstrate a superior capacity on the reduction of tHcy levels compared to the diffusive method. Moreover, the lower depletion of vitamin B12 by diffusive techniques could determine a higher reduction of folate levels, demonstrating the decisive role of the membrane performance in the treatment of this patients.
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Suplementos Dietéticos , Homocisteína/sangre , Fallo Renal Crónico/genética , Fallo Renal Crónico/terapia , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Diálisis Renal/métodos , Vitaminas/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Método Doble Ciego , Femenino , Ácido Fólico/uso terapéutico , Genotipo , Hemodiafiltración , Humanos , Fallo Renal Crónico/metabolismo , Masculino , Persona de Mediana Edad , Vitamina B 12/uso terapéutico , Adulto JovenRESUMEN
BACKGROUND: Methylmalonic aciduria with homocystinuria, cblC defect, is the most frequent disorder of vitamin B12 metabolism. CblC patients are commonly treated with a multidrug therapy to reduce metabolite accumulation and to increase deficient substrates. However the long-term outcome is often unsatisfactory especially in patients with early onset, with frequent progression of neurological and ocular impairment. Recent studies, have shown perturbation of cellular redox status in cblC. To evaluate the potential contribution of oxidative stress into the patophysiology of cblC defect, we have analyzed the in vivo glutathione metabolism in a large series of cblC deficient individuals. METHODS: Levels of different forms of glutathione were measured in lymphocytes obtained from 18 cblC patients and compared with age-matched controls. Furthermore, we also analyzed plasma cysteine and total homocysteine. RESULTS: We found an imbalance of glutathione metabolism in cblC patients with a significant decrease of total and reduced glutathione, along with a significant increase of different oxidized glutathione forms. CONCLUSIONS: These findings show a relevant in vivo disturbance of glutathione metabolism underlining the contribution of glutathione pool depletion to the redox imbalance in treated cblC patients. Our study may be helpful in addressing future research to better understanding the pathogenetic mechanism of the disease and in developing new therapeutic approaches, including the use of novel vitamin B12 derivatives.
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Errores Innatos del Metabolismo de los Aminoácidos/sangre , Cisteína/sangre , Glutatión/metabolismo , Homocisteína/sangre , Homocistinuria/sangre , Adolescente , Proteínas Portadoras/metabolismo , Estudios de Casos y Controles , Niño , Preescolar , Progresión de la Enfermedad , Femenino , Humanos , Lactante , Linfocitos/citología , Masculino , Oxidación-Reducción , Estrés Oxidativo , Oxidorreductasas , Oxígeno/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Vitamina B 12/metabolismo , Deficiencia de Vitamina B 12/congénitoRESUMEN
Non-alcoholic fatty liver disease (NAFLD) is a spectrum of metabolic abnormalities ranging from simple triglyceride accumulation in the hepatocytes to hepatic steatosis with inflammation, ballooning and fibrosis. It has been demonstrated that the pathogenesis of NAFLD involves increased oxidative stress, with consumption of the major cellular antioxidant, glutathione (GSH). Liver has a fundamental role in sulfur compound metabolism, although the data reported on plasma thiols status in NAFLD are conflicting. We recruited 63 NAFLD patients, and we analyzed all plasma thiols, such as homocysteine (Hcy), cysteine (Cys), cysteinylglycine (CysGly) and GSH, by high-performance liquid chromatography (HPLC) with fluorescence detection. Hcy, Cys and CysGly plasma levels increased in NAFLD patients (p < 0.0001); whereas GSH levels were decreased in NAFLD patients when compared to controls (p < 0.0001). On the contrary, patients with steatohepatitis exhibited lower levels of Hcy and Cys than subjects without. Furthermore, a positive correlation was found between Hcy and Cys and the presence of fibrosis in children with NAFLD. Taken together, these data demonstrated a defective hepatic sulfur metabolism in children with NAFLD, and that high levels of Hcy and Cys probably correlates with a pattern of more severe histological liver damage, due to mechanisms that require further studies.
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Cisteína/sangre , Homocisteína/sangre , Hígado/patología , Enfermedad del Hígado Graso no Alcohólico/sangre , Niño , Dipéptidos/sangre , Glutatión/sangre , Humanos , Masculino , Enfermedad del Hígado Graso no Alcohólico/patologíaRESUMEN
BACKGROUND: Genetically defined Leigh syndrome (LS) is a rare, fatal inherited neurodegenerative disorder that predominantly affects children. Although mitochondrial dysfunction has clearly been associated with oxidative stress, few studies have specifically examined Leigh syndrome patients' blood glutathione levels. In this study, we analyzed the balance between oxidized and reduced glutathione in lymphocytes of 10 patients with genetically confirmed LS and monitored the effects of glutathione status following 6 months of treatment with EPI-743, a novel redox therapeutic. METHODS: Lymphocytes were obtained from blood samples of 10 children with a genetically confirmed diagnosis of LS and in 20 healthy subjects. Total, reduced, oxidized and protein-bound glutathione levels were determined by HPLC analysis. Erythrocyte superoxide dismutase and glutathione peroxidase enzyme activities were measured by spectrophotometric assays. Plasma total thiols, carbonyl contents and malondialdehyde were assessed by spectrophotometric and fluorometric assays. RESULTS: A significant impairment of all glutathione forms was detected in patients, including a profound decrease of total and reduced glutathione (GSH) associated with high levels of all oxidized glutathione forms (GSSG+GS-Pro; OX). These findings negatively correlated with the glutathione peroxidase activity, which underwent a significant decrease in patients. After treatment with EPI-743, all patients showed a significant increase in reduced glutathione levels and 96% decrease of OX/GSH ratio. CONCLUSIONS: The data presented here strongly support glutathione as a "redox blood signature" in mitochondrial disorders and its use as a clinical trial endpoint in the development of mitochondrial disease therapies.
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Glutatión/sangre , Encefalomiopatías Mitocondriales/sangre , Ubiquinona/análogos & derivados , Adolescente , Estudios de Casos y Controles , Niño , Preescolar , Eritrocitos/enzimología , Glutatión Peroxidasa/metabolismo , Humanos , Lactante , Malondialdehído/sangre , Encefalomiopatías Mitocondriales/tratamiento farmacológico , Oxidación-Reducción , Superóxido Dismutasa/metabolismo , Resultado del Tratamiento , Ubiquinona/uso terapéuticoRESUMEN
Coenzyme Q(10) (CoQ(10)) is present in humans in both the reduced (ubiquinol, CoQ(10)H(2)) and oxidized (ubiquinone, CoQ(10)) forms. CoQ(10) is an essential cofactor in mitochondrial oxidative phosphorylation, and is necessary for ATP production. Total, reduced and oxidized CoQ(10) levels in skeletal muscle of 148 children were determined by HPLC coupled with electrochemical detection, and we established three level thresholds for total CoQ(10) in muscle. We defined as "severe deficiency", CoQ(10) levels falling in the range between 0.82 and 4.88 µmol/g tissue; as "intermediate deficiency", those ranging between 5.40 and 9.80 µmol/g tissue, and as "mild deficiency", the amount of CoQ(10) included between 10.21 and 19.10 µmol/g tissue. Early identification of CoQ(10) deficiency has important implications in children, not only for those with primary CoQ(10) defect, but also for patients with neurodegenerative disorders, in order to encourage earlier supplementation with this agent also in mild and intermediate deficiency.
Asunto(s)
Biomarcadores/metabolismo , Músculo Esquelético/metabolismo , Ubiquinona/análogos & derivados , Adolescente , Niño , Preescolar , Cromatografía Líquida de Alta Presión , Técnicas Electroquímicas , Femenino , Humanos , Lactante , Masculino , Enfermedades Neurodegenerativas/diagnóstico , Enfermedades Neurodegenerativas/metabolismo , Oxidación-Reducción , Valores de Referencia , Ubiquinona/deficiencia , Ubiquinona/metabolismoRESUMEN
PURPOSE: Paediatric obesity is a well-known risk factor for metabolic-associated fatty liver disease (MAFLD). The aim of this study was to evaluate the effects of laparoscopic sleeve gastrectomy (LSG) on the levels of total homocysteine (tHcy) and total glutathione (tGSH) plasma levels in children with MAFLD. MATERIAL AND METHODS: Twenty-four children with severe obesity who underwent LSG were included in the study. The metabolic parameters, systemic inflammatory markers, one-carbon metabolism products, ultrasound and histological improvement were evaluated at baseline (T0M) and after 12 months from LSG (T12M). RESULTS: The patients exhibited a significant amelioration of several metabolic parameters at T12M. A significant reduction of steatosis was observed at ultrasound (from 72.7% of moderate-severe grade to 0% severe steatosis), accompanied by a statistically significant improvement of ballooning, portal and lobular inflammation and fibrosis. A statistically significant decrease of tumour necrosis factor circulating levels was also observed (T0M median = 290.3, IQR = 281.0-317.0 pg/mL; T12M median = 260.4, IQR = 240.0-279.0 pg/mL; p < 0.0001). After 12 months from LSG, a significant increase of mean plasma levels of tHcy(T0M mean = 15.7 ± 4.1 µmol/L; T12M mean = 21.1 ± 9.3 µmol/L; p = 0.0146) was also observed. The increase of tHcy showed no causal link with the improvement of MAFLD-related inflammatory, metabolic and histological pattern. CONCLUSION: LSG in children with obesity induces an improvement of MAFLD-related metabolic derangement and liver damage, but also a mild hyperhomocysteinemia that should be avoided to prevent cardiovascular risk.
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Laparoscopía , Enfermedad del Hígado Graso no Alcohólico , Obesidad Mórbida , Obesidad Infantil , Niño , Gastrectomía/efectos adversos , Glutatión , Homocisteína , Humanos , Laparoscopía/efectos adversos , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Obesidad Mórbida/cirugía , Obesidad Infantil/complicaciones , Obesidad Infantil/cirugía , Resultado del TratamientoRESUMEN
A high-pressure liquid chromatography (HPLC) method with ultraviolet detection was developed for the measurement of plasma free and total tazobactam and piperacillin. This method is simple and fast, requiring only 11 min for the HPLC run and a sample preparation of about 11 min for total drugs and 10 min for free drugs. The procedure for the assay involves the treatment of plasma with acetonitrile for total drugs determination, and the use of a centrifugal filter device to deproteinize plasma for free drugs determination. The HPLC column, a Hypersil-ODS, was equilibrated with an eluent mixture composed of acetonitrile-potassium phosphate (pH 2.6). CVs for repeatability of tazobactam and piperacillin measurements ranged from 4.30 to 6.60; CVs for reproducibility ranged from 5.60 to 9.40. Mean analytical recoveries ranged from 100.4 to 103%. A linear relationship was obtained between peak area and drugs concentration in the range studied (0-62.5mg/L for tazobactam and 0-500mg/L for piperacillin). The equation for regression line were y=19x-1.4 for tazobactam and y=1.7x-0.9 for piperacillin; correlation coefficients were >0.999. The lower limit of quantitation (LLQ) for standard samples was about 0.12 mg/L for tazobactam and 0.49 mg/L for piperacillin, respectively. The lower limit of detection (LLD) was 0.06 mg/L for tazobactam and 0.24 mg/L for piperacillin. This HPLC assay for tazobactam and piperacillin is sensitive and accurate, and provides a reliable determination of both free and total tazobactam and piperacillin in human plasma, thus allowing the determination of these analytes in patients receiving tazocillin therapy.
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Antibacterianos/sangre , Cromatografía Líquida de Alta Presión/métodos , Ácido Penicilánico/análogos & derivados , Piperacilina/sangre , Humanos , Ácido Penicilánico/sangre , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , TazobactamRESUMEN
BACKGROUND: Hyperhomocysteinemia (hHcy) is a risk factor in the progression of chronic kidney disease (CKD). In type 2 diabetes (T2D), hHcy is strongly associated with increased risk of cardiovascular disease. Vitamin B12 and folic acid supplementation have been reported to lower homocysteine (tHcy) levels, but no data on plasma tHcy, cysteine (Cys), folate and vitamin B12 levels in T2D-CKD patients are reported. PROCEDURES: tHcy and Cys levels were analyzed in 178 T2D-CKD patients by high performance liquid chromatography (HPLC) with fluorescence detection. In addition, we determined folate and vitamin B12 levels using a chemiluminescence method. RESULTS: tHcy and Cys levels were increased in T2D patients, and this rise positively correlated with the CKD stage (P < 0.001). Folate levels were comparable to controls at various CKD stages, whereas vitamin B12 levels were lower, except at stage IV. We did not find any correlation between B-vitamins and levels of tHcy and Cys, regardless of the CKD stage. CONCLUSIONS: This is the first study reporting tHcy, Cys and B-vitamins status in T2D-CKD patients. Although limited to our cohort of 178 patients, our findings could be helpful in clarifying the conflicting literature regarding B-vitamins supplementation. Further studies are necessary before any Hcy-lowering therapy can be safely established in T2D-CKD subjects.
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Cisteína/sangre , Diabetes Mellitus Tipo 2/sangre , Ácido Fólico/sangre , Homocisteína/sangre , Insuficiencia Renal Crónica/sangre , Vitamina B 12/sangre , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Cromatografía Líquida de Alta Presión , Diabetes Mellitus Tipo 2/complicaciones , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Insuficiencia Renal Crónica/complicacionesRESUMEN
BACKGROUND/AIMS: Critically-ill patients often undergo continuous renal replacement therapy (CRRT) and need antimicrobial therapy. Piperacillin and tazobactam (Pip-Tzb) are cleared by CRRT. Our aim is to evaluate Pip-Tzb removal in an in-vitro-single-pool-model of continuous-veno-venous-hemofiltration (CVVH); we test a new method of Pip-Tzb administration during CRRT assuring constant levels of concentrations above the minimum inhibitory concentration (MIC). METHODS: In an in-vitro-single-pool-model of CVVH, two solutions (Protein-Free-Solution, PFS and Fresh-Frozen- Plasma, FFP) added with Pip-Tzb were tested for Pip-Tzb removal and adsorption. Then, to keep concentrations constantly above the MIC during CVVH, we add Pip-Tzb in the reinfusion bags. RESULTS: Pip-Tzb rapidly decreased than the MIC during CVVH. The adsorption was irrelevant in the test with FPS. Adding Pip-Tzb in the reinfusion bags of the CVVH system, we observed constant concentrations of Pip-Tzb over time. CONCLUSION: The association of Pip-Tzb is rapidly cleared with a real risk of inadequate dosages in patients undergoing CRRT. Adding Pip-Tzb in the reinfusion bags above the MIC, we obtained stability of concentrations during CVVH.
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Antibacterianos/administración & dosificación , Hemofiltración , Modelos Biológicos , Ácido Penicilánico/análogos & derivados , Antibacterianos/análisis , Antibacterianos/sangre , Técnicas In Vitro , Tasa de Depuración Metabólica , Ácido Penicilánico/administración & dosificación , Ácido Penicilánico/análisis , Ácido Penicilánico/sangre , Piperacilina/administración & dosificación , Piperacilina/análisis , Piperacilina/sangre , Combinación Piperacilina y TazobactamRESUMEN
BACKGROUND: Treatment with folic acid and vitamin B12 appears to be effective in lowering total plasma homocysteine (tHcy) concentrations, but whether vitamin B12 alone lowers tHcy in patients with normal vitamin B12 status is unknown. The aims of the present study were to explore the effect of individual supplementation with folic acid or vitamin B12 on tHcy concentrations in hemodialysis (HD) patients and to compare changes in tHcy concentrations with MTHFR genotype. METHODS: We recruited 200 HD patients (119 men) from the "Umberto I" Hospital (Frosinone, Italy) and the Dialysis Unit of University Hospital "Tor Vergata". These patients were randomized blindly into 2 groups of 100 each. Unfortunately, during the study, 36 patients in the first group and 16 in the second group died. The first group was treated initially with vitamin B12 for 2 months and with folic acid for a following 2 months. The second group was treated initially with folic acid and then with vitamin B12. Samples were drawn before administration of either, after the first and second periods, and again 2 months after treatment. RESULTS: The concentrations of tHcy decreased in both groups after the consecutive vitamin therapies, and the decrease was genotype-dependent. The decrease was greater for the T/T genotype (P <0.05) and was more significant when the treatment was started with folic acid (P <0.01). CONCLUSION: The alternating vitamin treatment demonstrated for the first time the importance of folate therapy and the secondary contribution of vitamin B12 in lowering tHcy in HD patients.