RESUMEN
To assess the discriminating power of multiple cerebrospinal fluid (CSF) biomarkers for Parkinson's disease (PD), we measured several proteins playing an important role in the disease pathogenesis. The activities of ß-glucocerebrosidase and other lysosomal enzymes, together with total and oligomeric α-synuclein, and total and phosphorylated tau, were thus assessed in CSF of 71 PD patients and compared to 45 neurological controls. Activities of ß-glucocerebrosidase, ß-mannosidase, ß-hexosaminidase, and ß-galactosidase were measured with established enzymatic assays, while α-synuclein and tau biomarkers were evaluated with immunoassays. A subset of PD patients (n = 44) was also screened for mutations in the ß-glucocerebrosidase-encoding gene (GBA1). In the PD group, ß-glucocerebrosidase activity was reduced (P < 0.05) and patients at earlier stages showed lower enzymatic activity (P < 0.05); conversely, ß-hexosaminidase activity was significantly increased (P < 0.05). Eight PD patients (18%) presented GBA1 sequence variations; 3 of them were heterozygous for the N370S mutation. Levels of total α-synuclein were significantly reduced (P < 0.05) in PD, in contrast to increased levels of α-synuclein oligomers, with a higher oligomeric/total α-synuclein ratio in PD patients when compared with controls (P < 0.001). A combination of ß-glucocerebrosidase activity, oligomeric/total α-synuclein ratio, and age gave the best performance in discriminating PD from neurological controls (sensitivity 82%; specificity 71%, area under the receiver operating characteristic curve = 0.87). These results demonstrate the possibility of detecting lysosomal dysfunction in CSF and further support the need to combine different biomarkers for improving the diagnostic accuracy of PD.
Asunto(s)
Glicósido Hidrolasas/líquido cefalorraquídeo , Enfermedad de Parkinson/líquido cefalorraquídeo , alfa-Sinucleína/líquido cefalorraquídeo , Adulto , Anciano , Femenino , Genotipo , Glucosilceramidasa/líquido cefalorraquídeo , Glucosilceramidasa/genética , Humanos , Inmunoensayo , Masculino , Persona de Mediana Edad , Mutación/genética , Enfermedad de Parkinson/genética , Estudios Prospectivos , Proteínas tau/líquido cefalorraquídeoRESUMEN
INTRODUCTION: This cross-sectional study was aimed at estimating the number of Italian incident cancer patients in 2020 eligible for, and respondent to, immune checkpoint inhibitors (ICI). METHODS: The study is based on publicly available data: the ICI approved until August 2022 by the Italian Medicines Agency (AIFA) with their specific indications and overall observed responses, rther details can be found in the Online Supplementary Materi cancer incidence estimates at 2020 and observed cancer deaths, and published papers with estimates on the frequency of different cancer stage/histology/markers etc. corresponding to AIFA authorizations. RESULTS: In the analyzed period, a total of seven ICI were authorized in Italy for 20 cancer types. The estimated number of ICI-eligible patients in 2020 was 48,400, 14.3% of those tumors (including skin epitheliomas) that may fit AIFA-indications, and 10.5% of all the incident malignant tumors, including skin epitheliomas. The number of patients who may benefit from ICI therapy was 24,052, 49.7% of the ICI-eligible ones, or 5.2% of the overall estimated incident cancers in 2020. CONCLUSIONS: In conclusion, although the number of ICI-eligible patients is a relatively small proportion of the yearly burden of cancers, about half of them may respond to ICI-treatment.
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Carcinoma , Neoplasias Cutáneas , Humanos , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Estudios Transversales , Italia/epidemiología , Estudios RetrospectivosRESUMEN
Most lysosomal storage diseases are caused by defects in genes encoding for acidic hydrolases. Deficiency of an enzyme involved in the catabolic pathway of N-linked glycans leads to the accumulation of the respective substrate and consequently to the onset of a specific storage disorder. Di-N-acetylchitobiase and core specific α1-6mannosidase represent the only exception. In fact, to date no lysosomal disease has been correlated to the deficiency of these enzymes. We generated di-N-acetylchitobiase-deficient mice by gene targeting of the Ctbs gene in murine embryonic stem cells. Accumulation of Man2GlcNAc2 and Man3GlcNAc2 was evaluated in all analyzed tissues and the tetrasaccharide was detected in urines. Multilamellar inclusion bodies reminiscent of polar lipids were present in epithelia of a scattered subset of proximal tubules in the kidney. Less constantly, enlarged Kupffer cells were observed in liver, filled with phagocytic material resembling partly digested red blood cells. These findings confirm an important role for lysosomal di-N-acetylchitobiase in glycans degradation and suggest that its deficiency could be the cause of a not yet described lysosomal storage disease.
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Acetilglucosaminidasa/metabolismo , Disacáridos/metabolismo , Enfermedades por Almacenamiento Lisosomal/enzimología , alfa-Manosidasa/metabolismo , Acetilglucosaminidasa/análisis , Acetilglucosaminidasa/deficiencia , Acetilglucosaminidasa/genética , Animales , Disacáridos/análisis , Células Madre Embrionarias , Marcación de Gen , Túbulos Renales Proximales/enzimología , Macrófagos del Hígado/enzimología , Hígado/enzimología , Lisosomas/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oligosacáridos/metabolismo , Oligosacáridos/orina , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Distribución Tisular , alfa-Manosidasa/análisis , beta-Glucosidasa/análisisRESUMEN
PURPOSE: The aim of this study was to estimate the Italian burden of incident breast cancer (BC) by subtypes, according to the distribution of hormonal receptor (HR) status and expression of human epidermal growth factor 2 (HER2). METHODS: Female breast cancers incidence in the Romagna Unit of the Emilia-Romagna registry (n. 10,711) were grouped into: HR+ /HER2-, HR+ /HER2+ , HR-/HER2+ , HR-/HER2- and missing, and by age: < 50, 50-69 and 70+ years. Data were compared with other published Italian population-bases series before using them for national estimates. We used national and regional numbers of expected breast cancers published by the Italian network of cancer registries considering the age- and geographic-specific variation of the Italian population. RESULTS: Overall, 70.7% of incident BC cases are expected to be HR+ /HER2-, 8.5% HR+ /HER2+ , 7.5% HR-/HER2-, 4.1% HR-/HER2+ and 9.3% missing. The global ranking is similar across age-groups but with age-specific differences. The proportion of missing was around 3-times lower than in the other Italian published population-based series and similar to the SEER one. In Italy, are estimated 38,841 HR+ /HER2- breast cancer cases, 4665 HR+ /HER2+ , 4098 HR-/HER2-, 2281 HR-/HER2+ , and 5092 not specified. Numbers by age-group were provided. CONCLUSIONS: The present estimates relied on high-quality population-based data and provide a clinically relevant information on the burden of breast cancer subtypes. These data will support the planning of therapy needs for oncologists, decision-makers, and all other stakeholders.
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Neoplasias de la Mama , Femenino , Humanos , Neoplasias de la Mama/metabolismo , Receptor ErbB-2/metabolismo , Sistema de Registros , Italia/epidemiología , Incidencia , Receptores de Progesterona/metabolismoRESUMEN
Deficiency in human lysosomal α-mannosidase (MAN2B1) results in α-mannosidosis, a lysosomal storage disorder; patients present a wide range of neurological, immunological, and skeletal symptoms caused by a multisystemic accumulation of mannose-containing oligosaccharides. Here, we describe the expression of recombinant MAN2B1 both transiently in Nicotiana benthamiana leaves and in the leaves and seeds of stably transformed N. tabacum plants. After purification from tobacco leaves, the recombinant enzyme was found to be N-glycosylated and localized in vacuolar compartments. In the fresh leaves of tobacco transformants, MAN2B1 was measured at 10,200 units/kg, and the purified enzyme from these leaves had a specific activity of 32-45 U/mg. Furthermore, tobacco-produced MAN2B1 was biochemically similar to the enzyme purified from human tissues, and it was internalized and processed by α-mannosidosis fibroblast cells. These results strongly indicate that plants can be considered a promising expression system for the production of recombinant MAN2B1 for use in enzyme replacement therapy.
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Nicotiana/metabolismo , alfa-Manosidasa/metabolismo , Línea Celular , Activación Enzimática , Pruebas de Enzimas , Fibroblastos/metabolismo , Glicosilación , Humanos , Inmunoprecipitación , Enfermedades por Deficiencia de Manosidasa/enzimología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Plásmidos/genética , Plásmidos/metabolismo , Protoplastos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Semillas/genética , Semillas/metabolismo , Nicotiana/genética , Transformación Genética , Vacuolas/metabolismo , alfa-Manosidasa/genética , alfa-Manosidasa/aislamiento & purificaciónRESUMEN
BACKGROUND: Prognostic factors in cutaneous melanoma are commonly evaluated by the Cox proportional hazard model. However, the interpretation of the effect of multiple variables is not straightforward. Classification and Regression Trees Analysis (CART), which allows a more friendly data evaluation, could be a valid integration of the message from Cox model. METHODS: The CART algorithm splits up data, creating a "tree" of groups of patients with different profiles for the risk of death. Results are easy to interpret in clinical practice. A total of 2692 patients with invasive cutaneous melanoma registered in Romagna (northern Italy) between 1993-2012 and followed-up until the end of 2013 were included. The Cox model and CART analysis were applied to sex, patient age, histological subtype, Breslow's tumor thickness, ulceration, site of disease, and Clark level. RESULTS: The CART analysis identified 15 categories which were collapsed into five classes with statistically different survival. The best prognostic group (10-year observed survival, 99.1%) included subjects with Breslow thickness ≤0.78 mm and age 16-81 years. The worst prognostic group (10-year observed survival, 35.8%) comprised subjects with thickness ≥3.75 mm and age 16-96 years. According to the Cox model, patient age, histological subtype, Breslow thickness, ulceration, and site of disease had a significant independent prognostic value. CONCLUSIONS: CART and Cox models provided consistent results. CART seemed friendlier in its interpretation and it could facilitate the communication of risk.
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Melanoma , Neoplasias Cutáneas , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Comunicación , Humanos , Melanoma/patología , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Neoplasias Cutáneas/patología , Adulto JovenRESUMEN
Mucolipidosis type III (MLIII) is an autosomal recessive disorder affecting lysosomal hydrolase trafficking. In a study of 10 patients from seven families with a clinical phenotype and enzymatic diagnosis of MLIII, six novel GNPTG gene mutations were identified. These included missense (p.T286M) and nonsense (p.W111X) mutations and a transition in the obligate AG-dinucleotide of the intron 8 acceptor splice site (c.610-2A>G). Three microdeletions were also identified, two of which (c.611delG and c.640_667del28) were located within the coding region whereas one (c.609+28_610-16del) was located entirely within intron 8. RT-PCR analysis of the c.610-2A>G transition demonstrated that the change altered splicing, leading to the production of two distinct aberrantly spliced forms, viz. the skipping of exon 9 (p.G204_K247del) or the retention of introns 8 and 9 (p.G204VfsX28). RT-PCR analysis, performed on a patient homozygous for the intronic deletion (c.609+28_610-16del), failed to detect any GNPTG RNA transcripts. To determine whether c.609+28_610-16del allele-derived transcripts were subject to nonsense-mediated mRNA decay (NMD), patient fibroblasts were incubated with the protein synthesis inhibitor anisomycin. An RT-PCR fragment retaining 43 bp of intron 8 was consistently detected suggesting that the 33-bp genomic deletion had elicited NMD. Quantitative real-time PCR and GNPTG western blot analysis confirmed that the homozygous microdeletion p.G204VfsX17 had elicited NMD resulting in failure to synthesize GNPTG protein. Analysis of the sequences surrounding the microdeletion breakpoints revealed either intrinsic repetitivity of the deleted region or short direct repeats adjacent to the breakpoint junctions. This is consistent with these repeats having mediated the microdeletions via replication slippage and supports the view that the mutational spectrum of the GNPTG gene is strongly influenced by the properties of the local DNA sequence environment.
Asunto(s)
Mucolipidosis/enzimología , Mucolipidosis/genética , Mutación/genética , Subunidades de Proteína/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Adolescente , Adulto , Empalme Alternativo/genética , Secuencia de Bases , Niño , Codón sin Sentido/genética , Femenino , Fibroblastos/enzimología , Fibroblastos/patología , Genotipo , Humanos , Masculino , Datos de Secuencia Molecular , Mutación Missense/genética , Sitios de Empalme de ARN/genética , Estabilidad del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de SecuenciaRESUMEN
BACKGROUND: Few studies have compared screen-detected (SD) breast cancer patients with symptomatic patients for the frequency and determinants of receipt of adjuvant systemic therapy according to accepted guidelines. METHODS: Depending on the date of diagnosis, adjuvant therapy guidelines from the 5th, 6th, and 7th St. Gallen International Conferences were used as standards to audit the treatment of 598 node-negative high-risk patients (59% SD) and 430 node-positive patients (40% SD) aged 50-69 years from an Italian cancer registry (1997-2001). Odds ratios (OR) and 95% confidence intervals (CI) were calculated using backward stepwise logistic regression models. RESULTS: Among node-negative high-risk patients, SD cancers were smaller (P = 0.000) and of lower grade (P = 0.003). Downgrading was generally from grade 3 to grade 2, with an increased proportion of patients placed in the high-risk category due to grade 2 alone. The total rates of adjuvant systemic therapy were similar (58 vs. 60%) whereas SD patients were less often treated according to the guidelines (34 vs. 45%; OR = 0.61; 95% CI, 0.44-0.86). After adjustment for tumour size and other weaker confounders, the OR was 0.99 (95% CI, 0.67-1.46). Among node-positive patients, the OR of receiving the standard adjuvant systemic therapy did not differ between SD and symptomatic cancers. CONCLUSIONS: SD cancers amplified the prognostic heterogeneity of node-negative high-risk patients. Their lower likelihood of being treated according to the guidelines was largely explained by their lower risk profile. No evidence was found to suggest that physicians held a priori assumptions about the relative biological indolence of SD cancers.
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Antineoplásicos/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/epidemiología , Anciano , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/patología , Quimioterapia Adyuvante , Detección Precoz del Cáncer , Femenino , Adhesión a Directriz , Humanos , Italia/epidemiología , Mamografía , Tamizaje Masivo , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Sistema de Registros , Análisis de SupervivenciaRESUMEN
The lysosomal enzyme di-N-acetylchitobiase hydrolyzes N-acetylglucosamine from the reducing-end of the N,N' diacetylchitobiose core of N-linked-oligosaccharides. The presence of chitobiase in the tissues of different species is probably responsible for differences in the structure of oligosaccharides accumulated in the lysosomal storage disease beta-mannosidosis. The disease has so far been described in humans, cats, cattle and goats. Low chitobiase activity has been observed in the tissues of ruminants and it has been hypothesized that in cattle this low level of expression is due to evolutionary changes in the promoter region. A cDNA encoding the mouse chitobiase has been isolated, sequenced and its identity confirmed by expression in COS-7 cells. Comparison of the mouse genomic sequence with the cDNA sequence revealed the presence of seven exons within the chitobiase gene. The gene spans about 15 kb and a single transcription initiation site was determined by 5'RACE. Chitobiase is differentially and ubiquitously expressed in mouse tissues as demonstrated by qRT-PCR analysis. Chitobiase is differentially expressed at lower levels in bovine tissues. In two bovine tissues (heart and muscle) mRNA was not detectable. Mouse and bovine promoters have been isolated and sequenced and their activities compared. The activity of the bovine promoter is very low and might explain the low activity of chitobiase observed in cattle.
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Acetilglucosaminidasa/genética , Lisosomas/enzimología , Ratones/genética , Animales , Secuencia de Bases , Células COS , Bovinos , Chlorocebus aethiops , Clonación Molecular , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , TransfecciónRESUMEN
Beta-mannosidosis (OMIM # 248510) is an autosomal-recessive lysosomal storage disorder caused by deficiency of the lysosomal enzyme beta-mannosidase (MANBA, E.C. 3.2.1.25). The disorder has been reported in goat, cattle and man. The human disorder is rare and only 20 cases in 16 families have been reported. We have sequenced the exons and exon-intron borders in a European patient with infantile onset of beta-mannosidosis. The patient was compound heterozygous for a silent mutation (c.375A>G) in exon 3 causing alternative splicing, and a missense mutation (c.1513T>C, p.Ser505Pro) in exon 12. The alternative splicing event deleted four nucleotides from the transcript and was predicted to result in premature termination of translation. In order to evaluate the consequence of the missense mutation, we inserted the human beta-mannosidase gene into an expression vector, performed site-directed mutagenesis and expressed the normal and mutant enzyme in COS-7 cells. We also included the previously reported beta-mannosidosis-associated missense mutations c.544C>T (p.Arg182Trp) and c.1175G>A (p.Gly392Glu), which were found in patients presenting a milder phenotype. Cells transfected with the wild-type construct showed a 33-fold increase in beta-mannosidase activity compared to mock-transfected cells, whereas cells transfected with the mutant constructs showed no detectable increase in activity. We propose that the milder phenotype described in some beta-mannosidosis patients with missense mutations in the MANBA gene is not due to residual beta-mannosidase activity, but rather caused by epigenetic and/or environmental factors.
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Mutación Missense , beta-Manosidasa/genética , beta-Manosidosis/enzimología , Empalme Alternativo , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Codón sin Sentido , Análisis Mutacional de ADN , Expresión Génica , Humanos , Población Blanca/genética , beta-Manosidasa/metabolismo , beta-Manosidosis/genéticaRESUMEN
Tissue infiltration is different in desmoid and fibroma tumours. Both produce high levels of transforming growth factor beta1 (TGFbeta1), which is related to extracellular matrix (ECM) accumulation which in turn regulates cell function and cell migration. Interactions between collagen, proteoglycans and cell surface fibronectin are involved in the assembly and functions of the ECM. As toremifene inhibits collagen and TGFbeta1 synthesis, we tested it in normal, desmoid and fibroma fibroblasts. We will report the changes in glycosaminoglycan (GAG) and collagen synthesis, TGFbeta1 activity, fibronectin mRNA expression and TGFbeta1 receptors after toremifene treatment in normal, fibroma and desmoid fibroblasts. We evaluated GAG and collagen synthesis with 3H-glucosamine and 3H-proline incorporation, TGFbeta1 activity with the ELISA method, TGFbeta1 receptor affinity with 125I-TGFbeta1 binding and total RNA with Northern blot analysis. GAG and collagen synthesis, TGFbeta1 activity and fibronectin levels were higher in fibroma and desmoid than normal fibroblasts. The increase was greater in desmoid than fibroma tumour cells. Toremifene treatment reduced GAG and collagen synthesis, TGFbeta1 activity and fibronectin levels in all cell cultures. The percentage reduction in GAG was similar in all cultures; the reduction in collagen synthesis and TGFbeta1 activity was the highest in desmoid fibroblasts. TGFbeta1 receptors were higher in fibroma and desmoid cells than controls. Toremifene reduced TGFbeta1 receptors only in desmoid fibroblasts, with no effect on the changes in type I, II, and III receptors. Our data show that toremifene modifies the ECM components that regulate cytokine activity and cell migration. The reduction in receptor number only in desmoid cells suggests that toremifene may reduce TGFbeta1's affinity for its receptors. Synthesis of a substance regulating protein kinase activity, which is directly involved in the link between TGFbeta1 and its receptors, cannot be excluded.
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Fibroblastos/metabolismo , Fibroma/metabolismo , Fibromatosis Agresiva/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteoglicanos/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Toremifeno/farmacología , Factor de Crecimiento Transformador beta1/metabolismo , Northern Blotting , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colágeno/biosíntesis , Fibroblastos/efectos de los fármacos , Fibronectinas/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Prolina/metabolismo , ARN/biosíntesis , ARN/aislamiento & purificación , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptor Tipo II de Factor de Crecimiento Transformador beta , Factor de Crecimiento Transformador beta1/antagonistas & inhibidoresRESUMEN
It is known that the neutral/cytosolic alpha-mannosidase (Man2c1) which can cleave alpha 1,2-, alpha 1,3-, and alpha 1,6-linked mannose residues, is stimulated by cobalt and is inhibited by furanose analogues swainsonine (SW) and 1,4-dideoxy-1,4-imino-d-mannitol (DIM). The enzyme is involved in the degradation of oligomannosides derived from dolichol intermediates and the degradation of newly synthesized glycoproteins. An immunological relationship has been demonstrated between the rat endoplasmic reticulum alpha-mannosidase and the cytosolic alpha-mannosidase. In fact antibodies raised against the soluble alpha-mannosidase recognized the membrane form of the ER alpha-mannosidase. A cDNA encoding the mouse cytosolic alpha-mannosidase was obtained by RZPD (Deutsches Ressourcenzentrum fur Genomforschung GmbH), Berlin, Germany. Comparison of the mouse genomic sequence with the cDNA sequence revealed the presence of 25 introns within the cytosolic alpha-mannosidase gene. The gene spans 11.5 kb from the major transcription initiation site to the RNA cleavage site. The transcription initiation site of mouse cytosolic alpha-mannosidase was mapped to 170 bases upstream of the ATG codon using 5' RACE. Northern blotting analysis revealed expression of a major transcript of 3.8 kb in all tissues examined. COS cells transfected with the cDNA showed a 20-fold increase in cytosolic alpha-mannosidase activity. This enzyme activity was stimulated by cobalt and inhibited by DIM and EDTA. Furthermore we demonstrated that the expressed enzyme was active towards the radiolabeled substrate Man9GlcNAc1 giving the final product Man5GlcNAc1 through the formation of Man8GlcNAc1 isomer C as intermediate.
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alfa-Manosidasa/genética , Regiones no Traducidas 3'/química , Regiones no Traducidas 5'/química , Animales , Secuencia de Bases , Células COS , Secuencia de Carbohidratos , Chlorocebus aethiops , Clonación Molecular , Citosol/enzimología , Exones , Intrones , Ratones , Datos de Secuencia Molecular , Distribución Tisular , alfa-Manosidasa/biosíntesisRESUMEN
Desmoid and fibroma tumours are characterized by cell proliferation, glycosaminoglycan and collagen fibre accumulation, high levels of transforming growth factor beta(1) (TGFbeta(1)) and different patterns of tissue infiltration. TGFbeta(1) is related to extracellular matrix (ECM) composition which, in turn, regulates cell functions and cell migration. In this study we report changes in cell proliferation, glycosaminoglycan (GAG) and collagen synthesis, TGFbeta(1) mRNA expression and fibronectin levels in normal, desmoid and fibroma fibroblast cultures before and after TGFbeta(1) stimulation. Our data showed cell proliferation, GAG and collagen synthesis, transforming growth factor beta(1) mRNA expression and fibronectin levels were significantly higher in desmoid than in fibroma cultures. TGFbeta(1) treatment had no effect on cell proliferation, but increased TGFbeta(1) mRNA expression, GAG, fibronectin and collagen synthesis in desmoid and fibroma fibroblasts. Its effects were more marked in desmoid cells. Fibronectin favours cell migration, while changes in GAG composition alter cell behaviour and ECM organization. In conclusion our data suggest that the different patterns of infiltration in desmoid and fibroma tumours are due to changes in ECM components and cell-ECM interactions which can be ascribed to altered TGFbeta(1) mRNA expression and TGFbeta(1) activity.
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Fibroblastos/metabolismo , Fibromatosis Agresiva/metabolismo , Leiomioma/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Northern Blotting , Adhesión Celular , Línea Celular , Movimiento Celular , Proliferación Celular , Colágeno/biosíntesis , Matriz Extracelular/metabolismo , Fibromatosis Agresiva/fisiopatología , Fibronectinas/metabolismo , Expresión Génica , Glicosaminoglicanos/biosíntesis , Humanos , Leiomioma/fisiopatología , ARN Mensajero/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismoRESUMEN
AIMS AND BACKGROUND: A registry-based cohort study of male patients with bladder cancer was conducted to determine the relative risk of second primary cancer of the prostate and kidney, the uni-/multivariate differences in relative risk according to patient characteristics, the cumulative risk by duration of the follow-up, and the prevalence:incidence ratio of prostate and kidney cancer cases detected in the first 6 months after the diagnosis of bladder cancer. METHODS: The complete case records of all male patients (n = 2025) diagnosed with bladder cancer between 1986 and 2002 were extracted from the database of the Romagna Cancer Registry: 1539 patients were eligible for analysis of the incidence of following prostate and kidney cancers, of the relative risk and the standardized incidence ratio specific for the time interval of follow-up. RESULTS: A total of 108 prostate cancer cases and 23 kidney cancer cases were observed during the follow-up. The relative risk of second primary cancer of the prostate and kidney was respectively 3.52 (95% CI, 2.89-4.25) and 3.90 (95% CI, 2.47-5.85). The absolute excess risk was 11.8 x 1000 for prostate cancer and 2.5 x 1000 for kidney cancer. The number of prevalent cases of prostate and kidney cancer detected was approximately 10 times greater than the expected number based on incidence rates from the general population. During the follow-up, incidence of prostate cancer stabilized at a level that was 3- to 4-fold greater than that expected. Despite fluctuations, a decrease was also observed for incidence of kidney cancer. CONCLUSIONS: In summary, our study showed the relatively constant high incidence of prostate and kidney cancers in bladder cancer patients over time. The possibility of subsequent cancer implies that an appropriate long surveillance is required. The pertinence depends on the duration of the follow-up as well as the degree of surveillance.
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Neoplasias Renales/epidemiología , Neoplasias Primarias Secundarias/epidemiología , Neoplasias de la Próstata/epidemiología , Neoplasias de la Vejiga Urinaria/epidemiología , Recolección de Datos , Estudios de Seguimiento , Humanos , Incidencia , Masculino , Estudios Retrospectivos , RiesgoRESUMEN
Hexavalent chromium compounds are well-documented human carcinogens. In vitro experiments show Cr (VI) induces cell death by apoptosis by activating p53 protein. The aim of this study was to evaluate Cr (VI)-induced apoptosis in a human bronchial epithelial cell line (BEAS-2B) and in a lymphoblastic leukemia cell line (MOLT-4). Cr (VI) caused a dose- and time-dependent increase in the apoptosis rate in both cell lines. Western blotting showed increased p53 protein expression in MOLT-4 cells, but not in BEAS-2B cells, after exposure to 0.5 and 3 muM hexavalent chromium for 12 hours and 4 hours, respectively. Apoptotic cell death induced by Cr (VI) was not decreased by pretreatment with caspase-3, -8, and -9 inhibitors. These preliminary results provide evidence of Cr (VI)-induced apoptosis, which deserves further investigation in occupationally exposed workers.
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Apoptosis , Carcinógenos Ambientales/efectos adversos , Cromo/efectos adversos , Proteína p53 Supresora de Tumor/metabolismo , Inhibidores de Caspasas , Línea Celular , Línea Celular Tumoral , Daño del ADN , Humanos , Leucemia Linfoide , Mucosa RespiratoriaRESUMEN
Mutation analysis performed on six Italian families with alpha-mannosidosis type II allowed the identification of five new mutations in the MAN2B1 gene: c.157G>T, c.562C>T, c.599A>T, c.293dupA, c.2402G>A (p.E53X, p.R188X, p.H200L, p.Y99VfsX61, p.G801D). Protein residues G801 and H200 are conserved among the four mammalian alpha-mannosidases cloned to date: human, cattle, cat and mouse. In vitro expression studies demonstrated that both missense mutations expressed no residual alpha-mannosidase activity indicating that they are disease-causing mutations. Modelling into the three-dimensional structure revealed that the p.H200L could involve the catalytic mechanism, whereas p.G801D would affect the correct folding of the enzyme.
Asunto(s)
Mutación Puntual , alfa-Manosidasa/genética , alfa-Manosidosis/genética , Animales , Catálisis , Gatos , Bovinos , Línea Celular , Codón sin Sentido , Consanguinidad , Análisis Mutacional de ADN , Humanos , Italia , Riñón , Lisosomas/enzimología , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación Missense , Reacción en Cadena de la Polimerasa , Conformación Proteica , Pliegue de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Especificidad de la Especie , alfa-Manosidasa/química , alfa-Manosidasa/deficiencia , alfa-Manosidasa/metabolismo , alfa-Manosidosis/clasificación , alfa-Manosidosis/enzimologíaRESUMEN
BACKGROUND: Desmoid tumour is a benign, non metastasising neoplasm characterised by an elevated deposition of organic macromolecules in the extracellular matrix (ECM). The matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases involved in the degradation of ECM macromolecules. The MMPs and their natural inhibitors (TIMPs) have been implicated in tumour growth, invasion and metastasis. In this study we provide evidence that the in vitro cultured cell line from desmoid tumour accumulates more collagen fibres in the ECM than healthy fibroblasts. METHODS: We investigated collagen accumulation by 3H-thymidine incorporation, MMP expression by substrate gel zymography and TIMP expression by Western blot analysis. RESULTS: Desmoid fibroblasts showed a reduction in MMP activity and an increase of type I and III collagen and TIMPs compared to normal fibroblasts. CONCLUSION: The increase in collagen in desmoid fibroblasts was due to inhibited collagen degradation (reduction of MMP activity) rather than to increased collagen synthesis. Adding toremifene, an anti-estrogen triphenylethylene derivate, to desmoid fibroblasts reduced collagen accumulation by decreasing mRNA expression and increasing collagen degradation.
Asunto(s)
Colágeno/análisis , Fibroblastos/metabolismo , Fibroblastos/patología , Fibromatosis Agresiva/metabolismo , Fibromatosis Agresiva/patología , Metaloproteinasas de la Matriz/metabolismo , Toremifeno/farmacología , Antineoplásicos Hormonales/farmacología , Línea Celular Tumoral , Colagenasas/metabolismo , Humanos , Inhibidores de la Metaloproteinasa de la Matriz , Metaloproteinasas de la Matriz/efectos de los fármacos , Procolágeno/análisis , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
We report the first newborn screening pilot study in an Italian region for four lysosomal disorders including Pompe disease, Gaucher disease, Fabry disease and mucopolysaccharidosis type 1. The screening has been performed using enzymatic assay on Dry Blood Spot on filter paper. A total of 3403 newborns were screened. One newborn showed a reduction of ß-glucosidase activity in leucocytes. Molecular analysis revealed a status of compound heterozygous for the panethnic mutation N370S and for the sequence variation E388K, not yet correlated to Gaucher disease onset. The functional consequences of the E388K replacement on ß-glucosidase activity were evaluated by in vitro expression, showing that the mutant protein retained 48% of wild type activity. Structural modeling predicted that the E388K replacement, localized to a surface of the enzyme, would change the local charges distribution which, in the native protein, displays an overwhelming presence of negative charges. However, the newborn, and a 4 year old sister showing the same genomic alterations, are currently asymptomatic. This pilot newborn screening for lysosomal diseases appears to be feasible and affordable to be extended to large populations. Moreover other lysosomal diseases for which a therapy is available or will be available, could be included in the screening.
Asunto(s)
Análisis Mutacional de ADN/métodos , Glucosilceramidasa/genética , Enfermedades por Almacenamiento Lisosomal/diagnóstico , Enfermedades por Almacenamiento Lisosomal/genética , Mutación , Tamizaje Neonatal/métodos , Femenino , Glucosilceramidasa/metabolismo , Células HEK293 , Humanos , Recién Nacido , Italia , Enfermedades por Almacenamiento Lisosomal/enzimología , Masculino , Proyectos PilotoRESUMEN
Recent studies have shown a genetic association between glucocerebrosidase deficiencies and Parkinson's disease (PD). To further explore this issue the activity of beta-glucocerebrosidase and the activities of other lysosomal enzymes, alpha-mannosidase, beta-mannosidase, beta-hexosaminidase, and beta-galactosidase have been evaluated in the cerebrospinal fluid (CSF) of PD patients. The activities of alpha-mannosidase, beta-mannosidase, beta-glucocerebrosidase, and beta-hexosaminidase were substantially decreased in the CSF of PD patients, while levels of beta-galactosidase were essentially identical to controls. This study indicates that in PD several lysosomal hydrolases have decreased activities, further supporting a possible link between pathophysiological mechanisms underlying PD and lysosomal hydrolases.
Asunto(s)
Hialuronoglucosaminidasa/líquido cefalorraquídeo , Enfermedad de Parkinson/líquido cefalorraquídeo , Anciano , Cromatografía por Intercambio Iónico/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estadísticas no ParamétricasRESUMEN
In the Crouzon's syndrome the cranial morphogenic processes are altered due to the early fusion of cranial sutures. We analysed the phenotype of cultured fibroblasts from normal subjects and from Crouzon patients with a specific fibroblast growth factor receptor 2 mutation resulting in a Cys 342 Tyr substitution within the third immunoglobulin domain. Crouzon fibroblasts differed from normal fibroblasts in their extracellular matrix macromolecule accumulation. In Crouzon fibroblasts glycosaminoglycans and fibronectin were decreased and type I collagen increased. As transforming growth factors beta (TGF beta) and basic fibroblasts growth factor (bFGF) together regulate extracellular matrix deposition, we evaluated TGF beta(1), TGF beta(3) and bFGF production by Crouzon and normal fibroblasts. TGF beta(1), TGFb(3) and bFGF levels were lower while TGF beta(1) mRNA transcripts were higher in Crouzon cells. As the increased TGF beta(1) gene expression did not translate into a parallel increase of secreted TGF beta(1), control of TGF beta(1) secretion may be mainly post-transcriptional. Furthermore, adding bFGF increased TGF beta(1) and TGF beta(3) secretion, suggesting the drop may be due to the altered signal transduction of bFGF. These innovative data suggest the in vitro differences between normal and Crouzon fibroblasts may be due to an imbalance in TGF beta and bFGF levels which alters the microenvironment where morphogenesis takes place.