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1.
Proc Natl Acad Sci U S A ; 118(22)2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34050011

RESUMO

CRISPR-Cas9 genome editing is a promising technique for clinical applications, such as the correction of disease-associated alleles in somatic cells. The use of this approach has also been discussed in the context of heritable editing of the human germ line. However, studies assessing gene correction in early human embryos report low efficiency of mutation repair, high rates of mosaicism, and the possibility of unintended editing outcomes that may have pathologic consequences. We developed computational pipelines to assess single-cell genomics and transcriptomics datasets from OCT4 (POU5F1) CRISPR-Cas9-targeted and control human preimplantation embryos. This allowed us to evaluate on-target mutations that would be missed by more conventional genotyping techniques. We observed loss of heterozygosity in edited cells that spanned regions beyond the POU5F1 on-target locus, as well as segmental loss and gain of chromosome 6, on which the POU5F1 gene is located. Unintended genome editing outcomes were present in ∼16% of the human embryo cells analyzed and spanned 4-20 kb. Our observations are consistent with recent findings indicating complexity at on-target sites following CRISPR-Cas9 genome editing. Our work underscores the importance of further basic research to assess the safety of genome editing techniques in human embryos, which will inform debates about the potential clinical use of this technology.


Assuntos
Blastocisto/metabolismo , Sistemas CRISPR-Cas , Edição de Genes , Células-Tronco Embrionárias Humanas/metabolismo , Perda de Heterozigosidade , Fator 3 de Transcrição de Octâmero , Linhagem Celular , Cromossomos Humanos Par 6/genética , Cromossomos Humanos Par 6/metabolismo , Humanos
2.
Epidemiol Prev ; 48(4-5): 333-346, 2024.
Artigo em Italiano | MEDLINE | ID: mdl-39301806

RESUMO

OBJECTIVES: to describe prevalence of disability in the population of the Agency for Health Protection of Milan (ATS Milan), integrating current administrative healthcare, socio-healthcare, and social data; to classify disability with a diagnosis into a predominant structural and functional category according to the International Classification of Functioning, Disability and Health (ICF), supplementing it with additional levels of detail. DESIGN: retrospective observational study. SETTING AND PARTICIPANTS: subjects residing in the territory of ATS Milan in the years from 2018 to 2022.  Main outcomes measures: prevalence of disability in the population of ATS Milan from 2018 to 2022; average annual costs since disability diagnosis of the entire population and stratified by the most common ICF classifications. RESULTS: the prevalence of disability ranges from 5.8% in 2018 to 8.4% in 2022. In general, women have a higher prevalence than men. However, there are significant differences in the gender distribution depending on the considered age group. The main disabilities (32.2%) affect the structures of the nervous system and mental functions, followed by disabilities identified solely by major prosthetic devices (9.4%) and sensory disabilities with alterations in sensory functions with the presence of a major device (5.2%). Analysis of average total annual per capita costs shows an upward trend with increasing years since the diagnosis. CONCLUSIONS: the definition of standardized tools, such as the selection from several available healthcare data provided by service suppliers, can be helpful in obtaining reliable data on the prevalence of disability in the population. This evidence can be useful in planning public health interventions to address the needs of this population. The work developed by ATS Milan has been carried out in alignment with the activities outlined in Mission 5 of the National Recovery and Resilience Plan (PNRR), in particular for the reform of disability legislation, which foresees the definition of standardized tools for the in-depth study of the epidemiological aspects of the phenomenon.


Assuntos
Algoritmos , Pessoas com Deficiência , Humanos , Itália/epidemiologia , Feminino , Masculino , Estudos Retrospectivos , Pessoas com Deficiência/estatística & dados numéricos , Pessoa de Meia-Idade , Adulto , Idoso , Prevalência , Adolescente , Bases de Dados Factuais , Criança , Adulto Jovem , Pré-Escolar , Avaliação da Deficiência , Lactente , Idoso de 80 Anos ou mais
3.
Mol Pharm ; 20(6): 2951-2965, 2023 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-37146162

RESUMO

Therapeutic proteins can be challenging to develop due to their complexity and the requirement of an acceptable formulation to ensure patient safety and efficacy. To date, there is no universal formulation development strategy that can identify optimal formulation conditions for all types of proteins in a fast and reliable manner. In this work, high-throughput characterization, employing a toolbox of five techniques, was performed on 14 structurally different proteins formulated in 6 different buffer conditions and in the presence of 4 different excipients. Multivariate data analysis and chemometrics were used to analyze the data in an unbiased way. First, observed changes in stability were primarily determined by the individual protein. Second, pH and ionic strength are the two most important factors determining the physical stability of proteins, where there exists a significant statistical interaction between protein and pH/ionic strength. Additionally, we developed prediction methods by partial least-squares regression. Colloidal stability indicators are important for prediction of real-time stability, while conformational stability indicators are important for prediction of stability under accelerated stress conditions at 40 °C. In order to predict real-time storage stability, protein-protein repulsion and the initial monomer fraction are the most important properties to monitor.


Assuntos
Anticorpos Monoclonais , Quimiometria , Humanos , Estabilidade Proteica , Anticorpos Monoclonais/química , Desdobramento de Proteína , Conformação Proteica , Estabilidade de Medicamentos
4.
Int J Mol Sci ; 24(13)2023 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-37445810

RESUMO

Intracellular pH (pHi) regulation is a challenge for the exocrine pancreas, where the luminal secretion of bicarbonate-rich fluid is accompanied by interstitial flows of acid. This acid-base transport requires a plethora of ion transporters, including bicarbonate transporters and the Na+/H+ exchanger isoform 1 (NHE1), which are dysregulated in Pancreatic Ductal Adenocarcinoma (PDAC). PDAC progression is favored by a Collagen-I rich extracellular matrix (ECM) which exacerbates the physiological interstitial acidosis. In organotypic cultures of normal human pancreatic cells (HPDE), parenchymal cancer cells (CPCs) and cancer stem cells (CSCs) growing on matrices reproducing ECM changes during progression, we studied resting pHi, the pHi response to fluxes of NaHCO3 and acidosis and the role of NHE1 in pHi regulation. Our findings show that: (i) on the physiological ECM, HPDE cells have the most alkaline pHi, followed by CSCs and CPCs, while a Collagen I-rich ECM reverses the acid-base balance in cancer cells compared to normal cells; (ii) both resting pHi and pHi recovery from an acid load are reduced by extracellular NaHCO3, especially in HPDE cells on a normal ECM; (iii) cancer cell NHE1 activity is less affected by NaHCO3. We conclude that ECM composition and the fluctuations of pHe cooperate to predispose pHi homeostasis towards the presence of NaHCO3 gradients similar to that expected in the tumor.


Assuntos
Acidose , Neoplasias , Humanos , Concentração de Íons de Hidrogênio , Bicarbonatos/metabolismo , Matriz Extracelular/metabolismo , Colágeno Tipo I , Ductos Pancreáticos/metabolismo , Células Epiteliais/metabolismo , Trocadores de Sódio-Hidrogênio
5.
Medicina (Kaunas) ; 59(6)2023 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-37374348

RESUMO

At the time of diagnosis, the vast majority of prostate carcinoma patients have a clinically localized form of the disease, with most of them presenting with low- or intermediate-risk prostate cancer. In this setting, various curative-intent alternatives are available, including surgery, external beam radiotherapy and brachytherapy. Randomized clinical trials have demonstrated that moderate hypofractionated radiotherapy can be considered as a valid alternative strategy for localized prostate cancer. High-dose-rate brachytherapy can be administered according to different schedules. Proton beam radiotherapy represents a promising strategy, but further studies are needed to make it more affordable and accessible. At the moment, new technologies such as MRI-guided radiotherapy remain in early stages, but their potential abilities are very promising.


Assuntos
Braquiterapia , Neoplasias da Próstata , Masculino , Humanos , Neoplasias da Próstata/radioterapia , Neoplasias da Próstata/patologia , Hipofracionamento da Dose de Radiação , Estudos Longitudinais
6.
Sensors (Basel) ; 22(24)2022 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-36560322

RESUMO

Breathing monitoring is crucial for evaluating a patient's health status. The technologies commonly used to monitor respiration are costly, bulky, obtrusive, and inaccurate, mainly when the user moves. Consequently, efforts have been devoted to providing new solutions and methodologies to overcome these limitations. These methods have several uses, including healthcare monitoring, measuring athletic performance, and aiding patients with respiratory diseases, such as COPD (chronic obtrusive pulmonary disease), sleep apnea, etc. Breathing-induced chest movements can be measured noninvasively and discreetly using inertial sensors. This research work presents the development and testing of an inertia-based chest band for breathing monitoring through a differential approach. The device comprises two IMUs (inertial measurement units) placed on the patient's chest and back to determine the differential inertial signal, carrying out information detection about the breathing activity. The chest band includes a low-power microcontroller section to acquire inertial data from the two IMUs and process them to extract the breathing parameters (i.e., RR-respiration rate; TI/TE-inhalation/exhalation time; IER-inhalation-to-exhalation time; V-flow rate), using the back IMU as a reference. A BLE transceiver wirelessly transmits the acquired breathing parameters to a mobile application. Finally, the test results demonstrate the effectiveness of the used dual-inertia solution; correlation and Bland-Altman analyses were performed on the RR measurements from the chest band and the reference, demonstrating a high correlation (r¯ = 0.92) and low mean difference (MD¯ = -0.27 BrPM (breaths per minute)), limits of agreement (LoA¯ = +1.16/-1.75 BrPM), and mean absolute error (MAE¯ = 1.15%). Additionally, the experimental results demonstrated that the developed device correctly measured the other breathing parameters (TI, TE, IER, and V), keeping an MAE of ≤5%. The obtained results indicated that the developed chest band is a viable solution for long-term breathing monitoring, both in stationary and moving users.


Assuntos
Síndromes da Apneia do Sono , Dispositivos Eletrônicos Vestíveis , Humanos , Respiração , Taxa Respiratória , Expiração
7.
Int J Mol Sci ; 23(10)2022 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-35628225

RESUMO

The cardiovascular benefit of statins is well established. However, only 20% of high-risk patients remain adequately adherent after 5 years of treatment. Among reasons for discontinuation, statin associated-muscle pain symptoms are the most prevalent. Aim of the present study was to evaluate the impact of high dose atorvastatin on skeletal muscle mitochondrial activity, aerobic and anaerobic exercise, and axonal excitability in a murine model of atherosclerosis. ApoE-/- mice were fed 12 weeks a high-fat high-cholesterol diet alone or containing atorvastatin (40 mg/Kg/day). Outcomes were the evaluation of muscle mitochondrial functionality, locomotion, grip test, and axonal excitability (compound action potential recording analysis of Aα motor propioceptive, Aß mechanoceptive and C nociceptive fibres). Atorvastatin led to a reduction in muscle mitochondrial biogenesis and mitochondrial ATP production. It did not affect muscular strength but led to a time-dependent motor impairment. Atorvastatin altered the responsiveness of mechanoceptive and nociceptive fibres, respectively, the Aß and C fibres. These findings point out to a mild sensitization on mechanical, tactile and pain sensitivity. In conclusion, although the prevalence of muscular side effects from statins may be overestimated, understanding of the underlying mechanisms can help improve the therapeutic approach and reassure adherence in patients needing-to-be-treated.


Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases , Doenças Musculares , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/farmacologia , Atorvastatina/farmacologia , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Locomoção , Camundongos , Músculo Esquelético , Doenças Musculares/induzido quimicamente
8.
Int J Mol Sci ; 23(21)2022 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-36361853

RESUMO

Vascular smooth muscle cells (VSMCs) are key participants in both early- and late-stage atherosclerosis and influence neighbouring cells possibly by means of bioactive molecules, some of which are packed into extracellular vesicles (EVs). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is expressed and secreted by VSMCs. This study aimed to unravel the role of PCSK9 on VSMCs-derived EVs in terms of content and functionality. EVs were isolated from human VSMCs overexpressing human PCSK9 (VSMCPCSK9-EVs) and tested on endothelial cells, monocytes, macrophages and in a model of zebrafish embryos. Compared to EVs released from wild-type VSMCs, VSMCPCSK9-EVs caused a rise in the expression of adhesion molecules in endothelial cells and of pro-inflammatory cytokines in monocytes. These acquired an increased migratory capacity, a reduced oxidative phosphorylation and secreted proteins involved in immune response and immune effector processes. Concerning macrophages, VSMCPCSK9-EVs enhanced inflammatory milieu and uptake of oxidized low-density lipoproteins, whereas the migratory capacity was reduced. When injected into zebrafish embryos, VSMCPCSK9-EVs favoured the recruitment of macrophages toward the site of injection. The results of the present study provide evidence that PCSK9 plays an inflammatory role by means of EVs, at least by those derived from smooth muscle cells of vascular origin.


Assuntos
Vesículas Extracelulares , Pró-Proteína Convertase 9 , Animais , Humanos , Pró-Proteína Convertase 9/metabolismo , Músculo Liso Vascular/metabolismo , Peixe-Zebra/metabolismo , Células Endoteliais/metabolismo , Miócitos de Músculo Liso/metabolismo , Vesículas Extracelulares/metabolismo
9.
J Sci Food Agric ; 102(11): 4500-4513, 2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-35122271

RESUMO

BACKGROUND: There is increasing consumer demand for olive oil to be traceable. However, genotype, environmental factors, and stage of maturity, all affect the flavor and composition of both the olives and olive oil. Few studies have included all three variables. Key metabolites include lipids, phenolics, and a wide range of volatile organic compounds (VOCs), which provide the olives and oil with their characteristic flavor. Here we aim to identify markers that are able to discriminate between cultivars, that can identify growth location, and can discriminate stages of fruit maturity. 'Nocellara messinese' and 'Carolea' olive fruits were grown at three locations differing in altitude in Calabria, Italy, and harvested at three stages of maturity. Oil was analyzed from the two most mature stages. RESULTS: Nine and 20 characters discriminated all fruit and oil samples respectively, and relative abundance of two fatty acids distinguished all oils. Whole VOC profiles discriminated among the least mature olives, and oil VOC profiles discriminated location and cultivar at both stages. Three VOCs putatively identified as hexanal, methyl acetate, and 3-hexen-1-ol differentiated all samples of oils from the most mature fruit stage. CONCLUSION: The results confirm that interactions of location, cultivar and fruit maturity stage are critical for the overall pattern of aroma compounds, and identify potential markers of commercial relevance. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.


Assuntos
Olea , Compostos Orgânicos Voláteis , Frutas/química , Olea/química , Azeite de Oliva/química , Fenóis/análise , Compostos Orgânicos Voláteis/química
10.
Medicina (Kaunas) ; 58(9)2022 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-36143894

RESUMO

In recent years, the growing use of ART (assisted reproductive techniques) has led to a progressive improvement of protocols; embryo freezing is certainly one of the most important innovations. This technique is selectively offered as a tailored approach to reduce the incidence of multiple pregnancies and, most importantly, to lower the risk of developing ovarian hyperstimulation syndrome when used in conjunction with an ovulation-triggering GnRH antagonist. The increase in transfer cycles with frozen embryos made it possible to study the effects of the technique in children thus conceived. Particularly noteworthy is the increase in macrosomal and LGA (large for gestational age) newborns, in addition to a decrease in SGA (small for gestational age) and LBW (low birth weight) newborns. The authors aimed to outline a broad-ranging narrative review by summarizing and elaborating on the most important evidence regarding the neonatal outcome of children born from frozen embryos and provide information on the medium and long-term follow- up of these children. However, given the relatively recent large-scale implementation of such techniques, further studies are needed to provide more conclusive evidence on outcomes and implications.


Assuntos
Criopreservação , Transferência Embrionária , Criança , Transferência Embrionária/efeitos adversos , Transferência Embrionária/métodos , Feminino , Seguimentos , Hormônio Liberador de Gonadotropina , Humanos , Recém-Nascido , Gravidez , Técnicas de Reprodução Assistida
11.
Pain Pract ; 22(4): 487-496, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35014151

RESUMO

BACKGROUND AND AIMS: This living and systematic review aimed to provide an updated summary of the available evidence on pain undertreatment prevalence in patients with cancer; correlations with some potential determinants and confounders were also carried out. MATERIALS AND METHODS: We updated a systematic review published on 2014, including observational and experimental studies reporting the use of the pain management index (PMI) in adults with cancer and pain, from 2014 to 2020. We conducted searches in PubMed/MEDLINE, Embase, and Google Scholar. We performed univariate and multivariable regression analyses to describe the relationship between PMI and a list of potential explanatory variables. RESULTS: Twenty new papers were identified, yielding a total sample size of 66 studies. The proportion of patients classified as undertreated according to the year of study publication shows a higher decrease from 1994 to 2013 (-13% as relative change) than the most recent years 2014-2020 (-11%). The quality of the included studies has increased over the years (from 80% to 93%). At the multivariable analysis, a statistically significant relationship was confirmed between undertreatment and the year of the publication of the study and with a low-medium economic level of the countries, where the studies were conducted. DISCUSSION: Despite the improvement when compared to the period 1994-2000, still about 40% of the cases identified received an analgesic treatment inadequate to the intensity of pain, according to the PMI. Despite its intrinsic limitations, PMI continues to be widely used, and it could allow a continuous monitoring of pain management across a different mix of studies and patients.


Assuntos
Analgésicos , Neoplasias , Adulto , Analgésicos/uso terapêutico , Humanos , Neoplasias/complicações , Neoplasias/tratamento farmacológico , Dor/tratamento farmacológico , Dor/epidemiologia , Dor/etiologia , Manejo da Dor , Medição da Dor
12.
Am J Pathol ; 190(11): 2226-2236, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32798443

RESUMO

In a condition of dysfunctional visceral fat depots, as in the case of obesity, alterations in adipokine levels may be detrimental for the cardiovascular system. The proinflammatory leptin and resistin adipokines have been described as possible links between obesity and atherosclerosis. The present study was aimed at evaluating whether proprotein convertase subtilisin/kexin type 9 (PCSK9), a key regulator of low-density lipoprotein metabolism, is induced by leptin and resistin through the involvement of the inflammatory pathway of STAT3. In HepG2 cells, leptin and resistin up-regulated PCSK9 gene and protein expression, as well as the phosphorylation of STAT3. Upon STAT3 silencing, leptin and resistin lost their ability to activate PCSK9. The knockdown of STAT3 did not affect the expression of leptin and resistin receptors or that of PCSK9. The analysis of the human PCSK9 promoter region showed that the two adipokines raised PCSK9 promoter activity via the involvement of a sterol regulatory element motif. In healthy males, a positive association between circulating leptin and PCSK9 levels was found only when the body mass index was <25 kg/m2. In conclusion, this study identified STAT3 as one of the molecular regulators of leptin- and resistin-mediated transcriptional induction of PCSK9.


Assuntos
Regulação Enzimológica da Expressão Gênica , Leptina/metabolismo , Pró-Proteína Convertase 9/biossíntese , Resistina/metabolismo , Fator de Transcrição STAT3/metabolismo , Regulação para Cima , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Células Hep G2 , Humanos , Leptina/genética , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologia , Pró-Proteína Convertase 9/genética , Resistina/genética , Elementos de Resposta , Fator de Transcrição STAT3/genética
13.
J Enzyme Inhib Med Chem ; 36(1): 1258-1267, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34107824

RESUMO

COVID-19, a pandemic disease caused by a viral infection, is associated with a high mortality rate. Most of the signs and symptoms, e.g. cytokine storm, electrolytes imbalances, thromboembolism, etc., are related to mitochondrial dysfunction. Therefore, targeting mitochondrion will represent a more rational treatment of COVID-19. The current work outlines how COVID-19's signs and symptoms are related to the mitochondrion. Proper understanding of the underlying causes might enhance the opportunity to treat COVID-19.


Assuntos
Tratamento Farmacológico da COVID-19 , COVID-19/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Antivirais/química , Antivirais/farmacologia , COVID-19/metabolismo , Humanos , Mitocôndrias/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/patogenicidade
14.
Int J Mol Sci ; 22(4)2021 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-33671549

RESUMO

Tumor cell invasion depends largely on degradation of the extracellular matrix (ECM) by protease-rich structures called invadopodia, whose formation and activity requires the convergence of signaling pathways engaged in cell adhesion, actin assembly, membrane regulation and ECM proteolysis. It is known that ß1-integrin stimulates invadopodia function through an invadopodial p(T567)-ezrin/NHERF1/NHE1 signal complex that regulates NHE1-driven invadopodia proteolytic activity and invasion. However, the link between ß1-integrin and this signaling complex is unknown. In this study, in metastatic breast (MDA-MB-231) and prostate (PC-3) cancer cells, we report that integrin-linked kinase (ILK) integrates ß1-integrin with this signaling complex to regulate invadopodia activity and invasion. Proximity ligation assay experiments demonstrate that, in invadopodia, ILK associates with ß1-integrin, NHE1 and the scaffold proteins p(T567)-ezrin and NHERF1. Activation of ß1-integrin increased both invasion and invadopodia activity, which were specifically blocked by inhibition of either NHE1 or ILK. We conclude that ILK integrates ß1-integrin with the ECM proteolytic/invasion signal module to induce NHE1-driven invadopodial ECM proteolysis and cell invasion.


Assuntos
Integrina beta1/metabolismo , Podossomos/metabolismo , Podossomos/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Humanos , Masculino , Células PC-3 , Fosfoproteínas/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo
15.
Int J Mol Sci ; 22(8)2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923431

RESUMO

Human atherosclerotic plaque contains smooth muscle cells (SMCs) negative for the contractile phenotype (α-smooth muscle actin) but positive for proprotein convertase subtilisin/kexin type 9 (PCSK9). Thus, we generated rat SMCs which overexpressed human PCSK9 (SMCsPCSK9) with the aim of investigating the role of PCSK9 in the phenotype of SMCs. PCSK9 overexpression in SMCsPCSK9 led to a significant downregulation of the low-density lipoprotein receptor (Ldlr) as well as transgelin (Sm22α), a marker of the contractile phenotype. The cell proliferation rate of SMCsPCSK9 was significantly faster than that of the control SMCs (SMCspuro). Interestingly, overexpression of PCSK9 did not impact the migratory capacity of SMCs in response to 10% FCS, as determined by Boyden's chamber assay. Expression and activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr) was significantly increased in the presence of PCSK9, both in SMCPCSK9 and after treatment with recombinant PCSK9. The transcriptional activity of sterol regulatory element-binding protein (SREBP) was also increased in the presence of PSCK9, suggesting a direct role of PCSK9 in the control of SRE-responsive genes, like HMGCR. We also observed that cholesterol biosynthesis is elevated in SMCPCSK9, potentially explaining the increased proliferation observed in these cells. Finally, concentration-dependent experiments with simvastatin demonstrated that SMCsPCSK9 were partially resistant to the antiproliferative and antimigratory effect of this drug. Taken together, these data further support a direct role of PCSK9 in proliferation, migration, and phenotypic changes in SMCs-pivotal features of atherosclerotic plaque development. We also provide new evidence on the role of PCSK9 in the pharmacological response to statins-gold standard lipid-lowering drugs with pleiotropic action.


Assuntos
Anticolesterolemiantes/farmacologia , Proliferação de Células , Miócitos de Músculo Liso/metabolismo , Pró-Proteína Convertase 9/metabolismo , Sinvastatina/farmacologia , Animais , Movimento Celular , Células Cultivadas , Colesterol/metabolismo , Feminino , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Pró-Proteína Convertase 9/genética , Ratos , Receptores de LDL
16.
Epidemiol Prev ; 45(3): 196-204, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34212701

RESUMO

OBJECTIVES: to assess the potential of a new algorithm based on current healthcare databases to identify potential cases of malformation, particularly chromosomal anomalies associated with terminations of pregnancy. DESIGN: retrospective observational study. SETTING AND PARTICIPANTS: Registry of Congenital Anomalies of Milan, live births, still births, and termination of pregnancies for fetal anomalies from 2012 to 2016, detected by using current healthcare data. MAIN OUTCOME MEASURES: prevalence between 2012 and 2016 of congenital malformations recorded by Milan's Registry of Congenital Anomalies, with particular regard to chromosomal anomaly trends. Variation in the percentage of malformations detected from terminations of pregnancy. RESULTS: prevalence of malformations increased from 270 in 2012 to 283 per 10,000 in 2016; specifically, chromosomal abnormalities increased from 35 to 51 per 10,000 births. The algorithm detected a greater proportion of anomalies associated with therapeutic abortion, especially with respect to chromosomal anomalies, with an increase from 57.7% in 2012 to 75.8% in 2016 (test for trend p=0.002). CONCLUSIONS: the proposed algorithm identified a greater number of chromosomal anomalies that caused termination of pregnancy and may be applied to existing Italian registries to evaluate the quality of healthcare services, in particular with regard to the effectiveness of prenatal trisomy screening policies. The algorithm may also be used where no active surveillance systems are present, as well as in epidemiological studies, to assess environmental impact on congenital anomalies.


Assuntos
Algoritmos , Aberrações Cromossômicas , Anormalidades Congênitas , Diagnóstico Pré-Natal , Anormalidades Congênitas/diagnóstico , Anormalidades Congênitas/epidemiologia , Feminino , Humanos , Itália/epidemiologia , Gravidez , Prevalência , Sistema de Registros , Reprodutibilidade dos Testes , Estudos Retrospectivos
17.
J Autoimmun ; 112: 102466, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32414606

RESUMO

The ready availability of human blood makes it the first choice for immuno-monitoring. However, this has been largely confined to static metrics, particularly resting T cell phenotypes. Conversely, dynamic assessments have mostly relied on cell stimulation in vitro which is subject to multiple variables. Here, immunodynamic insights from the peripheral blood are shown to be obtainable by applying a revised approach to cell-cycle analysis. Specifically, refined flow cytometric protocols were employed, assuring the reliable quantification of T cells in the S-G2/M phases of the cell-cycle (collectively termed "T Double S" for T cells in S-phase in Sanguine: in short "TDS" cells). Without protocol refinement, TDS could be either missed, as most of them layed out of the conventional lymphocyte gates, or confused with cell doublets artefactually displaying high DNA-content. To illustrate the nature of TDS cells, and their relationship to different immunodynamic scenarios, we examined them in healthy donors (HD); infectious mononucleosis (IM) patients versus asymptomatic EBV+ carriers; and recently-diagnosed T1D patients. TDS were reproducibly more abundant among CD8+ T cells and a defined subset of T-regulatory CD4+ T cells, and were substantially increased in IM and a subset of T1D patients. Of note, islet antigen-reactive TDS cell frequencies were associated with an aggressive T cell effector phenotype, suggesting that peripheral blood can reflect immune events within tissues in T1D, and possibly in other organ-specific autoimmune diseases. Our results suggest that tracking TDS cells may provide a widely applicable means of gaining insight into ongoing immune response dynamics in a variety of settings, including tissue immunopathologies where the peripheral blood has often not been considered insightful.


Assuntos
Pontos de Checagem do Ciclo Celular/imunologia , Monitorização Imunológica/métodos , Linfócitos T/imunologia , Animais , Citometria de Fluxo/métodos , Humanos , Camundongos , Camundongos Transgênicos
18.
Bioconjug Chem ; 31(1): 123-129, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31794200

RESUMO

Antibody-drug conjugates (ADCs) are an emerging class of biopharmaceutical products for oncology, with the cytotoxic pyrrolobenzodiazepine (PBD) family of "warheads" well-established in the clinic. While PBDs offer high potency, they are also characterized by their hydrophobicity, which can make formulation of the ADC challenging. Several approaches have been investigated to improve the physicochemical properties of PBD-containing ADCs, and herein a supramolecular approach was explored using cucurbit[8]uril (CB[8]). The ability of CB[8] to simultaneously encapsulate two guests was exploited to incorporate a 12-mer polyethylene glycol harboring a methyl viologen moiety at one terminus (MV-PEG12), together with a PBD harboring an indole moiety at the C2' position (SG3811). This formulation approach successfully introduced a hydrophilic PEG to mask the hydrophobicity of SG3811, improving the physical stability of the ADC while avoiding any loss of potency related to chemical modification.


Assuntos
Benzodiazepinas/química , Hidrocarbonetos Aromáticos com Pontes/química , Imidazóis/química , Imunoconjugados/química , Pirróis/química , Estabilidade de Medicamentos , Interações Hidrofóbicas e Hidrofílicas , Polietilenoglicóis/química
19.
Mol Pharm ; 17(2): 426-440, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31790599

RESUMO

Therapeutic protein candidates should exhibit favorable properties that render them suitable to become drugs. Nevertheless, there are no well-established guidelines for the efficient selection of proteinaceous molecules with desired features during early stage development. Such guidelines can emerge only from a large body of published research that employs orthogonal techniques to characterize therapeutic proteins in different formulations. In this work, we share a study on a diverse group of proteins, including their primary sequences, purity data, and computational and biophysical characterization at different pH and ionic strength. We report weak linear correlations between many of the biophysical parameters. We suggest that a stability comparison of diverse therapeutic protein candidates should be based on a computational and biophysical characterization in multiple formulation conditions, as the latter can largely determine whether a protein is above or below a certain stability threshold. We use the presented data set to calculate several stability risk scores obtained with an increasing level of analytical effort and show how they correlate with protein aggregation during storage. Our work highlights the importance of developing combined risk scores that can be used for early stage developability assessment. We suggest that such scores can have high prediction accuracy only when they are based on protein stability characterization in different solution conditions.


Assuntos
Anticorpos Monoclonais/química , Descoberta de Drogas/métodos , Imunoglobulina G/química , Interferon alfa-2/química , Desdobramento de Proteína , Albumina Sérica Humana/química , Transferrina/química , Sequência de Aminoácidos , Armazenamento de Medicamentos , Humanos , Concentração de Íons de Hidrogênio , Concentração Osmolar , Agregados Proteicos , Estabilidade Proteica , Projetos de Pesquisa , Solubilidade
20.
Eur Heart J Suppl ; 22(Suppl L): L53-L56, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33239979

RESUMO

Compelling evidence has emerged from epidemiological and Mendelian randomization analyses relative to the causality of lipoprotein(a) [Lp(a)] in atherosclerotic cardiovascular diseases (ASCVD), being elevated Lp(a) a strong risk factor regardless of the reduction of LDL-C achieved by statins. So far, no specific available agent can lower Lp(a) to the extent required to achieve a cardiovascular (CV) benefit, i.e. approximately 100 mg/dL. The most recent outcomes trial FOURIER with evolocumab showed that a 25 nmol/L (12 mg/dL) reduction in Lp(a) corresponded to a 15% decrement in the relative risk of cardiovascular disease. The ODYSSEY OUTCOMES trial with alirocumab has been the first demonstrating that a reduction in Lp(a) associates with less major adverse cardiovascular events (MACE), i.e. hazard ratio: 0.994 per 1 mg/dL decrement in Lp(a). The Lp(a) lowering effect driven by PCSK9 inhibition was confirmed in carriers of PCSK9 loss-of-function mutations in which Lp(a) and oxPL-apoB levels were decreased compared to non-carriers as was for a slight larger number of apo(a) Kringle IV repeats. Although PCSK9 inhibitors are not able to decrease Lp(a) to the extent required to achieve a CV benefit, their use has led to a higher discontinuation rate in lipoprotein apheresis in patients with progressive ASCVD and high plasma Lp(a).

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