RESUMO
Neuroinflammatory conditions in the central nervous system (CNS) are implicated in the pathogenesis of several neuroimmune disorders such as acquired demyelinating syndromes, autoimmune encephalopathies, acute or chronic bacterial and viral CNS infections as well as multiple sclerosis (MS) [...].
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Doenças Neuroinflamatórias , Humanos , Doenças Neuroinflamatórias/imunologia , Animais , Esclerose Múltipla/terapia , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Esclerose Múltipla/tratamento farmacológico , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Sistema Nervoso Central/imunologia , InflamaçãoRESUMO
Dolutegravir (DTG) is one of the most prescribed antiretroviral drugs for treating people with HIV infection, including women of child-bearing potential or pregnant. Nonetheless, neuropsychiatric symptoms are frequently reported. Early reports suggested that, probably in relation to folic acid (FA) shortage, DTG may induce neural tube defects in infants born to women taking the drug during pregnancy. Subsequent reports did not definitively confirm these findings. Recent studies in animal models have highlighted the association between DTG exposure in utero and congenital anomalies, and an increased risk of neurologic abnormalities in children exposed during in utero life has been reported. Underlying mechanisms for DTG-related neurologic symptoms and congenital anomalies are not fully understood. We aimed to deepen our knowledge on the neurodevelopmental effects of DTG exposure and further explore the protective role of FA by the use of zebrafish embryos. We treated embryos at 4 and up to 144 h post fertilization (hpf) with a subtherapeutic DTG concentration (1 µM) and observed the disruption of the anterior-posterior axis and several morphological malformations in the developing brain that were both prevented by pre-exposure (2 hpf) and rescued by post-exposure (10 hpf) with FA. By whole-mount in situ hybridization with riboprobes for genes that are crucial during the early phases of neurodevelopment (ntl, pax2a, ngn1, neurod1) and by in vivo visualization of the transgenic Tg(ngn1:EGFP) zebrafish line, we found that DTG induced severe neurodevelopmental defects over time in most regions of the nervous system (notochord, midbrain-hindbrain boundary, eye, forebrain, midbrain, hindbrain, spinal cord) that were mostly but not completely rescued by FA supplementation. Of note, we observed the disruption of ngn1 expression in the dopaminergic regions of the developing forebrain, spinal cord neurons and spinal motor neuron projections, with the depletion of the tyrosine hydroxylase (TH)+ dopaminergic neurons of the dorsal diencephalon and the strong reduction in larvae locomotion. Our study further supports previous evidence that DTG can interfere with FA pathways in the developing brain but also provides new insights regarding the mechanisms involved in the increased risk of DTG-associated fetal neurodevelopmental defects and adverse neurologic outcomes in in utero exposed children, suggesting the impairment of dopaminergic pathways.
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Ácido Fólico , Compostos Heterocíclicos com 3 Anéis , Oxazinas , Piperazinas , Piridonas , Peixe-Zebra , Animais , Compostos Heterocíclicos com 3 Anéis/farmacologia , Ácido Fólico/metabolismo , Oxazinas/farmacologia , Piridonas/farmacologia , Piperazinas/farmacologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Defeitos do Tubo Neural/induzido quimicamente , Neurogênese/efeitos dos fármacos , FemininoRESUMO
As most new medications, Cabotegravir (CAB) was recently approved as an antiretroviral treatment of HIV infection without in-depth safety information on in utero exposure. Although no developmental toxicity in rats and rabbits was reported, recent studies demonstrated that CAB decreases pluripotency of human embryonic stem cells. CAB exposure effects during development were assessed in zebrafish embryos by the Fish Embryo Toxicity test after exposure at subtherapeutic concentrations up to 25× the human Cmax. Larvae behavior was assessed by the light-dark locomotion test. The expression of factors involved in neurogenesis was evaluated by whole-mount in situ hybridization. CAB did not cause gross morphological defects at low doses, although pericardial edema, uninflated swim bladder, decreased heartbeats, growth delay, and decreased hatching rate were observed at the highest concentrations. Decreased locomotion was observed even at the subtherapeutic dose, suggesting alterations of nervous system integrity. This hypothesis was supported by the observation of decreased expression of crucial factors involved in early neuronal differentiation in diencephalic and telencephalic dopaminergic areas, midbrain/hindbrain boundary, and craniofacial ganglia. These findings support CAB effects on neurogenesis in zebrafish embryos and suggest long-term follow-up of exposed infants to provide data on drug safety during pregnancy.
Assuntos
Infecções por HIV , Poluentes Químicos da Água , Humanos , Animais , Coelhos , Ratos , Peixe-Zebra , Embrião não Mamífero , Frequência Cardíaca , Larva , Poluentes Químicos da Água/toxicidadeRESUMO
In the past, one of the most widely used non-nucleoside reverse transcriptase inhibitors (NNRTI) in first-line antiretroviral therapy (ART) of HIV infection was efavirenz (EFV), which is already used as a cost-effective treatment in developing countries due to its efficacy, tolerability, and availability. However, EFV also demonstrates several adverse effects, like hepatotoxicity, altered lipid profile, neuropsychological symptoms, and behavioral effects in children after in utero exposure. In 2018, another NNRTI, doravirine (DOR), was approved due to its similar efficacy but better safety profile. Preclinical safety studies demonstrated that DOR is not genotoxic and exhibits no developmental toxicity or effects on fertility in rats. Zebrafish (Danio rerio) embryos have been widely accepted as a vertebrate model for pharmacological and developmental studies. We used zebrafish embryos as an in vivo model to investigate the developmental toxicity of DOR compared to EFV. After exposure of the embryos to the drugs from the gastrula stage up to different developmental stages (30 embryos for each arm, in three independent experiments), we assessed their survival, morphology, hatching rate, apoptosis in the developing head, locomotion behavior, vasculature development, and neutral lipid distribution. Overall, DOR showed a better safety profile than EFV in our model. Therapeutic and supra-therapeutic doses of DOR induced very low mortality [survival rates: 92, 90, 88, 88, and 81% at 1, 5, 10, 25, and 50 µM, respectively, at 24 h post fecundation (hpf), and 88, 85, 88, 89, and 75% at the same doses, respectively, at 48 hpf] and mild morphological alterations compared to EFV exposure also in the sub-therapeutic ranges (survival rates: 80, 77, 69, 63, and 44% at 1, 5, 10, 25, and 50 µM, respectively, at 24 hpf and 72, 70, 63, 52, and 0% at the same doses, respectively, at 48 hpf). Further, DOR only slightly affected the hatching rate at supra-therapeutic doses (97, 98, 96, 87, and 83% at 1, 5, 10, 25, and 50 µM, respectively, at 72 hpf), while EFV already strongly reduced hatching at sub-therapeutic doses (83, 49, 11, 0, and 0% at 1, 5, 10, 25, and 50 µM, respectively, at the same time endpoint). Both DOR at therapeutic doses and most severely EFV at sub-therapeutic doses enhanced apoptosis in the developing head during crucial phases of embryo neurodevelopment and perturbed the locomotor behavior. Furthermore, EFV strongly affected angiogenesis and disturbed neutral lipid homeostasis even at sub-therapeutic doses compared to DOR at therapeutic concentrations. Our findings in zebrafish embryos add further data confirming the higher safety of DOR with respect to EFV regarding embryo development, neurogenesis, angiogenesis, and lipid metabolism. Further studies are needed to explore the molecular mechanisms underlying the better pharmacological safety profile of DOR, and further human studies are required to confirm these results in the zebrafish animal model.
Assuntos
Fármacos Anti-HIV , Infecções por HIV , Humanos , Criança , Animais , Ratos , Infecções por HIV/tratamento farmacológico , Fármacos Anti-HIV/farmacologia , Peixe-Zebra , Inibidores da Transcriptase Reversa/toxicidade , Lipídeos/farmacologia , Embrião não MamíferoRESUMO
Cognitive decline of aging is modulated by chronic inflammation and comorbidities. In people with HIV-infection (PWH) it may also be affected by HIV-induced inflammation, lifestyle and long-term effects of antiretroviral therapies (ART). The role of genetics in the susceptibility to HIV-associated neurocognitive disorders (HAND) is not fully understood. Here we explored the possible relations among variants in 3 genes involved in inflammation and neurodegenerative disorders (APOE: ε2/ε3/ε4; HFE: H63D; C9ORF72: hexanucleotide expansions ≥ 9 repeats), cognitive/functional impairment (MiniMental State Examination MMSE, Clock Drawing Test CDT, Short Physical Performance Battery SPPB), comorbidities and HIV-related variables in a cohort of > 50 years old PWH (n = 60) with at least 10 years efficient ART. Patients with diabetes or hypertension showed significantly lower MMSE (p = .031) or SPPB (p = .010) scores, respectively, while no relations between HIV-related variables and cognitive/functional scores were observed. Patients with at least one APOEε3 allele had higher CDT scores (p = .019), APOEε2/ε4 patients showing the lowest scores in all tests. Patients with HFE-H63D variant showed more frequently hypertriglyceridemia (p = .023) and those harboring C9ORF72 expansions > 9 repeats had higher CD4+-cell counts (p = .032) and CD4% (p = .041). Multiple linear regression analysis computed to verify possible associations among cognitive/functional scores and all variables further suggested positive association between higher CDT scores and the presence of at least one APOEε3 allele (2,2; 95% CI [0,03 0,8]; p = .037), independent of other variables, although the model did not reach the statistical significance (p = .14). These data suggest that in PWH on efficient ART cognitive abilities and physical performances may be partly associated with comorbidities and genetic background. However, further analyses are needed to establish whether they could be also dependent and influenced by comorbidities and genetic background.
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Apolipoproteínas E/genética , Infecções por HIV , Desempenho Físico Funcional , Proteína C9orf72/genética , Genótipo , Infecções por HIV/complicações , Infecções por HIV/epidemiologia , Infecções por HIV/genética , Proteína da Hemocromatose/genética , Humanos , Inflamação , Pessoa de Meia-Idade , Testes Neuropsicológicos , Projetos PilotoRESUMO
Persistent inflammation, despite anti-retroviral therapy (ART), is an independent predictor of mortality and comorbidities in HIV infection. Multiple factors, including lifestyle and chronic viral coinfections, may contribute. Several of these factors are also associated with a chronic inflammation in the general population. Little is known about the degree to which these factors influence inflammation in HIV infection, particularly within the first year of ART. The purpose of this study was to distinguish the effects of factors (gender, body mass index, cholesterol and triglyceride levels, smoke habit and cytomegalovirus seropositivity), known to contribute to inflammation, on inflammation biomarkers over the first year of ART in HIV-infected patients. Linear mixed model analysis revealed significant biomarker decreases [soluble CD14 (s-CD14), soluble CD163 (s-CD163) and D-dimer (DD)], or increases [C Reactive Protein (CRP) and interleukin-6 (IL-6)] over time in the whole cohort, differences in most categories (genders for IL-6, smoke habit for s-CD14, cytomegalovirus infection for s-CD163 and IL-6) and in some category × time interactions [gender for interleukin-7 (IL-7)], cytomegalovirus infection for s-CD14 and cholesterol levels for s-CD14 and Tumor Necrosis Factor α (TNF-α)]. This explorative longitudinal study suggests further investigations on targeting inflammation pathophysiology in HIV-infected patients on ART.
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Terapia Antirretroviral de Alta Atividade , Índice de Massa Corporal , Infecções por Citomegalovirus/sangue , Infecções por HIV/tratamento farmacológico , Inflamação/sangue , Lipídeos/sangue , Caracteres Sexuais , Fumar/sangue , Adulto , Idoso , Biomarcadores/sangue , Quimioterapia Combinada , Feminino , Infecções por HIV/sangue , Infecções por HIV/virologia , Hábitos , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
A cytokine storm, autoimmune features and dysfunctions of myeloid cells significantly contribute to severe coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Genetic background of the host seems to be partly responsible for severe phenotype and genes related to innate immune response seem critical host determinants. The C9orf72 gene has a role in vesicular trafficking, autophagy regulation and lysosome functions, is highly expressed in myeloid cells and is involved in immune functions, regulating the lysosomal degradation of mediators of innate immunity. A large non-coding hexanucleotide repeat expansion (HRE) in this gene is the main genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), both characterized by neuroinflammation and high systemic levels of proinflammatory cytokines, while HREs of intermediate length, although rare, are more frequent in autoimmune disorders. C9orf72 full mutation results in haploinsufficiency and intermediate HREs seem to modulate gene expression as well and impair autophagy. Herein, we sought to explore whether intermediate HREs in C9orf72 may be a risk factor for severe COVID-19. Although we found intermediate HREs in only a small portion of 240 patients with severe COVID-19 pneumonia, the magnitude of risk for requiring non-invasive or mechanical ventilation conferred by harboring intermediate repeats >10 units in at least one C9orf72 allele was more than twice respect to having shorter expansions, when adjusted for age (odds ratio (OR) 2.36; 95% confidence interval (CI) 1.04-5.37, p = 0.040). The association between intermediate repeats >10 units and more severe clinical outcome (p = 0.025) was also validated in an independent cohort of 201 SARS-CoV-2 infected patients. These data suggest that C9orf72 HREs >10 units may influence the pathogenic process driving more severe COVID-19 phenotypes.
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Proteína C9orf72/genética , COVID-19/patologia , Repetições de Microssatélites , Adulto , Fatores Etários , Idoso , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , COVID-19/genética , COVID-19/virologia , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Fatores de Risco , SARS-CoV-2/isolamento & purificação , Índice de Gravidade de DoençaRESUMO
The use of combination anti-retroviral therapy (cART) correlates with longer and healthier life and with nearly normal life expectancy in people living with HIV. However, cART does not completely restore health. Chronic immune activation and inflammation persist in treated patients and have been described as predictors for clinical events and mortality in HIV-infected patients. Limited information is available on the impact of the various cART regimens on inflammation/immunoactivation. The aim of this work was to explore the impact of elvitegravir, dolutegravir, raltegravir (integrase strand transfer inhibitors, INSTIs) and atazanavir (protease inhibitor, PI) on several soluble markers of immune activation and inflammation during the first year of effective combination anti-retroviral therapy (cART). We conducted an observational retrospective cohort study in HIV-infected cART-naïve patients who initiated an INSTI or atazanavir regimen between March 2015 and February 2016 and a serum sample was available at baseline, 6 and 12â¯months after initiation. We compared the trend of D-Dimer, TNF- α, IL-2, IL-6, IL-7, IL-10, CCL4/MIP1-ß, CCL5/RANTES, s-CD14, s-CD163, hs-CRP levels among the 4 arms of treatment. Percentage of variation from baseline was also measured for all markers. A total of 36 patients were included. We observed heterogeneous modifications in inflammation markers among arms. In particular, we noted that EVG have significant negative effect on s-CD14, hs-CRP, IL-6 and D-Dimer in respect to other INSTIs and this different effect occurs mainly during the first 6â¯months of cART. IL-7 values increased in the three arms with INSTIs (significantly only in EGV, 159.8%, pâ¯=â¯0.0003) and decreased significantly in patients on PI (-48.96%; pâ¯=â¯0.04) over the period. In conclusion, our results provide further data on changes of inflammatory marker levels, especially for the new INSTIs. Our data show that among INSTIs, EVG seems to have a worse impact on inflammation.
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Fármacos Anti-HIV/uso terapêutico , Citocinas/sangue , Quimioterapia Combinada , Infecções por HIV/tratamento farmacológico , Inibidores de Integrase de HIV/uso terapêutico , Adulto , Sulfato de Atazanavir/uso terapêutico , Feminino , HIV-1/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Humanos , Inflamação/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Oxazinas/uso terapêutico , Piperazinas/uso terapêutico , Piridonas/uso terapêutico , Quinolonas/uso terapêutico , Raltegravir Potássico/uso terapêuticoRESUMO
The anti-malarial drugs chloroquine (CQ) and primarily the less toxic hydroxychloroquine (HCQ) are currently used to treat autoimmune diseases for their immunomodulatory and anti-thrombotic properties. They have also been proposed for the treatment of several viral infections, due to their anti-viral effects in cell cultures and animal models, and, currently, for the treatment of coronavirus disease 2019 (COVID-19), the pandemic severe acute respiratory syndrome caused by coronavirus 2 (Sars-Cov-2) infection that is spreading all over the world. Although in some recent studies a clinical improvement in COVID-19 patients has been observed, the clinical efficacy of CQ and HCQ in COVID-19 has yet to be proven with randomized controlled studies, many of which are currently ongoing, also considering pharmacokinetics, optimal dosing regimen, therapeutic level and duration of treatment and taking into account patients with different severity degrees of disease. Here we review what is currently known on the mechanisms of action of CQ and HCQ as anti-viral, anti-inflammatory and anti-thrombotic drugs and discuss the up-to-date experimental evidence on the potential mechanisms of action of CQ/HCQ in Sars-Cov2 infection and the current clinical knowledge on their efficacy in the treatment of COVID-19 patients. Given the role of iron in several human viral infections, we also propose a different insight into a number of CQ and HCQ pharmacological effects, suggesting a potential involvement of iron homeostasis in Sars-Cov-2 infection and COVID-19 clinical course.
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Betacoronavirus/efeitos dos fármacos , Cloroquina/farmacologia , Cloroquina/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Homeostase/efeitos dos fármacos , Hidroxicloroquina/farmacologia , Hidroxicloroquina/uso terapêutico , Ferro/metabolismo , Pneumonia Viral/tratamento farmacológico , Antivirais/farmacologia , Antivirais/uso terapêutico , COVID-19 , Infecções por Coronavirus/metabolismo , Humanos , Pandemias , Pneumonia Viral/metabolismo , SARS-CoV-2 , Tratamento Farmacológico da COVID-19RESUMO
Anemia is the main extra-gastrointestinal symptom in inflammatory bowel diseases (IBDs). Interleukin-6 (IL-6) and other cytokines are secreted and act in the microenvironment of the small intestine mucous membrane of IBD patients. Iron is essential for multiple cell functions and its homeostasis is regulated by the hepcidin-ferroportin axis. Hepcidin (HEPC) is mainly produced by the liver in response to iron needs but is also an acute phase protein. During inflammation, hepcidin is upregulated by IL-6 and is responsible for iron compartmentalization within cells, in turn causing anemia of inflammation. Tissues other than liver can produce hepcidin in response to inflammatory stimuli, in order to decrease iron efflux at a local level, then acting in an autocrine-paracrine manner. In IBDs and, in particular, in celiac disease (CeD), IL-6 might trigger the expression, upregulation and secretion of hepcidin in the small intestine, reducing iron efflux and exacerbating defective iron absorption. 7-Hydroxymatairesinol (7-HMR) belongs to the family of lignans, polyphenolic compounds produced by plants, and has nutraceutical antioxidant, anti-inflammatory and estrogenic properties. In this mini-review we revise the role of inflammation in IBDs and in particular in CeD, focusing our attention on the close link among inflammation, anemia and iron metabolism. We also briefly describe the anti-inflammatory and estrogenic activity of 7-HMR contained in foods that are often consumed by CeD patients. Finally, considering that HEPC expression is regulated by iron needs, inflammation and estrogens, we explored the hypothesis that 7-HMR consumption could ameliorate anemia in CeD using Caco-2 cells as bowel model. Further studies are needed to verify the regulation pathway through which 7-HMR may interfere with the local production of HEPC in bowel.
Assuntos
Anemia/metabolismo , Anti-Inflamatórios/farmacologia , Doença Celíaca/metabolismo , Hepcidinas/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Ferro/metabolismo , Lignanas/farmacologia , Animais , Antioxidantes/farmacologia , Células CACO-2 , Doença Celíaca/dietoterapia , Doença Celíaca/imunologia , Citocinas/metabolismo , Grão Comestível/química , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Doenças Inflamatórias Intestinais/dietoterapia , Doenças Inflamatórias Intestinais/imunologia , Lignanas/química , Lignanas/metabolismoRESUMO
7-Hydroxymatairesinol (7-HMR) is a plant lignan abundant in various concentrations in plant foods. The objective of this study was to test HMRLignan™, a purified form of 7-HMR, and the corresponding Picea abies extract (total extract P. abies; TEP) as dietary supplements on a background of a high-fat diet (HFD)-induced metabolic syndrome in mice and in the 3T3-L1 adipogenesis model. Mice, 3 weeks old, were fed a HFD for 60 d. Subgroups were treated with 3 mg/kg body weight 7-HMR (HMRLignan™) or 10 mg/kg body weight TEP by oral administration. 7-HMR and TEP limited the increase in body weight (-11 and -13 %) and fat mass (-11 and -18 %) in the HFD-fed mice. Epididymal adipocytes were 19 and -12 % smaller and the liver was less steatotic (-62 and -65 %). Serum lipids decreased in TEP-treated mice (-11 % cholesterol, -23 % LDL and -15 % TAG) and sugar metabolism was ameliorated by both lignan preparations, as shown by a more than 70 % decrease in insulin secretion and insulin resistance. The expression of several metabolic genes was modulated by the HFD with an effect that was reversed by lignan. In 3T3-L1 cells, the 7-HMR metabolites enterolactone (ENL) and enterodiol (END) showed a 40 % inhibition of cell differentiation accompanied by the inhibited expression of the adipogenic genes PPARγ, C/EBPα and aP2. Furthermore, END and ENL caused a 10 % reduction in TAG uptake in HEPA 1-6 hepatoma cells. In conclusion, 7-HMR and TEP reduce metabolic imbalances typical of the metabolic syndrome and obesity in male mice, whereas their metabolites inhibit adipogenesis and lipid uptake in vitro.
Assuntos
Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Dieta Hiperlipídica , Lignanas/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Síndrome Metabólica/tratamento farmacológico , Picea/química , Células 3T3-L1 , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacologia , 4-Butirolactona/uso terapêutico , Adipogenia/efeitos dos fármacos , Adipogenia/genética , Tecido Adiposo/citologia , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Fármacos Antiobesidade/farmacologia , Fármacos Antiobesidade/uso terapêutico , Suplementos Nutricionais , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/metabolismo , Expressão Gênica , Resistência à Insulina , Lignanas/uso terapêutico , Lipídeos/sangue , Masculino , Síndrome Metabólica/sangue , Síndrome Metabólica/etiologia , Síndrome Metabólica/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/sangue , Obesidade/etiologia , Obesidade/metabolismo , Obesidade/prevenção & controle , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêuticoRESUMO
BACKGROUND: Anemia is frequent during HIV infection and is predictive of mortality. Although cART has demonstrated to reduce its prevalence, several patients still experience unresolved anemia. We aimed to characterize iron homeostasis and inflammation in HIV-infected individuals with mild anemia in relation to cART. METHODS: In this retrospective cohort study, HIV-infected patients with mild anemia, CD4+ cells > 200/mm3 at baseline, maintaining virological response for 12 months after cART starting were selected within the Standardized Management of Antiretroviral Therapy Cohort (MASTER) cohort. Several inflammation and immune activation markers and iron homeostasis indexes were measured in stored samples, obtained at cART initiation (T0) and 12 months later (T1). Patients were grouped on the basis of hemoglobin values at T1: group A (> 13 g/dl) and B (< 13 g/dl). Wilcoxon rank sum test was used to compare biomarker values. Pearson correlation coefficients were calculated for all variables. RESULTS: cART improved CD4+ and CD8+ cell counts and their ratio, but this effect was significant only in group A. Only these patients had mild iron deficiency at T0 and showed higher transferrin and lower percentage of transferrin saturation than patients of group B, but differences disappeared with cART. cART decreased inflammation in all patients, but group B had higher levels of all markers than group A, reaching statistical significance only for IL-8 values at T1 (16 vs 2.9 pg/ml; p = 0.017). Hepcidin and IL-6 levels did not show significant differences between groups. Hemoglobin levels both at T0 and T1 did not correlate with any marker. CONCLUSIONS: Baseline mild anemia in HIV-infected patients cannot always be resolved with durable efficient cART, possibly due to residual inflammation or immune activation rather than unbalanced iron homeostasis. Further research is needed on cytokine profiling to understand the mechanisms that induce anemia in HIV with suppressive cART.
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Anemia/tratamento farmacológico , Fármacos Anti-HIV/uso terapêutico , Biomarcadores/sangue , Infecções por HIV/sangue , Infecções por HIV/tratamento farmacológico , Homeostase , Inflamação/patologia , Ferro/metabolismo , Adulto , Anemia/sangue , Anemia/complicações , Fármacos Anti-HIV/farmacologia , Demografia , Infecções por HIV/complicações , Infecções por HIV/epidemiologia , Homeostase/efeitos dos fármacos , Humanos , Inflamação/sangue , Inflamação/complicações , Pessoa de Meia-IdadeRESUMO
PURPOSE: Conventional methods used to identify BRCA1/2 germline mutations in hereditary cancers are time-consuming and expensive, due to the large size of the genes. The recent introduction of next generation sequencing (NGS) benchtop platforms is a great promise, which is rapidly revolutionizing genetic screening in diagnostic and clinical applications. We recently transferred our methodology for routine BRCA1/2 mutation screening (denaturing High Performance Liquid Chromatography plus Sanger sequencing) to the Ion Torrent PGM platform with the Ion Ampliseq BRCA1 and BRCA2 panel and tested the performance of the system. METHODS: We first validated the NGS approach in a cohort of 33 patients who had previously undergone genetic diagnosis in our laboratory by conventional methods. Then, we tested 29 newly diagnosed and uncharacterized patients by NGS, and Sanger sequencing was used to confirm results from the NGS platform. RESULTS: In the validation cohort, all previously identified single nucleotide variants, insertions and deletions (also composed of multiple bases and within complex homopolymeric stretches) were identified by NGS in their correct zygosity status except for variants in a complex multinucleotide region within intron 7 of BRCA1 gene. NGS approach was further able to identify previously undetected variants. In the prospective cohort, almost all (99.3%) called variants were confirmed by Sanger. In both cohorts, in addition to the false positive (31) and false negative (110) results in the intron 7 of BRCA1 gene, the NGS method detected 10 false positives, that were solved by Sanger. CONCLUSIONS: The Ion Torrent PGM NGS approach in BRCA1/2 germline mutation identification is highly sensitive, easy to use, faster and cheaper than traditional approaches. Therefore, according to other recently published works, we highly recommend this system for routine diagnostic testing on BRCA1/2 genes, along with Sanger confirmation of the called variants, and support the usefulness of the approach also in other routine genetic analysis.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Mutação em Linhagem Germinativa , Análise de Sequência de DNA/métodos , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Feminino , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Íntrons , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/genética , Estudos Prospectivos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Deleção de SequênciaRESUMO
Although large expansions of the non-coding GGGGCC repeat in C9orf72 gene are clearly defined as pathogenic for Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD), intermediate-length expansions have also been associated with those and other neurodegenerative diseases. Intermediate-length allele sizing is complicated by intrinsic properties of current PCR-based methodologies, in that somatic mosaicism could be suspected. We designed a protocol that allows the exact sizing of intermediate-length alleles, as well as the identification of large expansions.
Assuntos
Alelos , Reação em Cadeia da Polimerase/métodos , Proteínas/genética , Proteína C9orf72 , Eletroforese em Gel de Ágar , Genótipo , HumanosRESUMO
Trace concentration of EDs (endocrine disrupting compounds) in water bodies caused by wastewater treatment plant effluents is a recognized problem for the health of aquatic organisms and their potential to affect human health. In this paper we show that continuous exposure of male mice from early development to the adult life (140 days) to unrestricted drinking of wastewater collected from a municipal sewage treatment plant, is associated with an increased adipose deposition and weight gain during adulthood because of altered body homeostasis. In parallel, bisphenol A (BPA) at the administration dose of 5 µg/kg/body weight, shows an increasing effect on total body weight and fat mass. In vitro, a solid phase extract (SPE) of the wastewater (eTW), caused stimulation of 3T3-L1 adipocyte differentiation at dilutions of 0.4 and 1 % in the final culture medium which contained a concentration of BPA of 40 nM and 90 nM respectively. Pure BPA also promoted adipocytes differentiation at the concentration of 50 and 80 µM. BPA effect in 3T3-L1 cells was associated to the specific activation of the estrogen receptor alpha (ERα) in undifferentiated cells and the estrogen receptor beta (ERß) in differentiated cells. BPA also activated the Peroxisome Proliferator Activated Receptor gamma (PPARγ) upregulating a minimal 3XPPARE luciferase reporter and the PPARγ-target promoter of the aP2 gene in adipose cells, while it was not effective in preadipocytes. The pure estrogen receptor agonist diethylstilbestrol (DES) played an opposite action to that of BPA inhibiting PPARγ activity in adipocytes, preventing cell differentiation, activating ERα in preadipocytes and inhibiting ERα and ERß regulation in adipocytes. The results of this work show that the drinking of chemically-contaminated wastewater promotes fat deposition in male mice and that EDs present in sewage are likely responsible for this effect through a nuclear receptor-mediated mechanism.
Assuntos
Adipócitos/efeitos dos fármacos , Tecido Adiposo/efeitos dos fármacos , Disruptores Endócrinos/toxicidade , Águas Residuárias/toxicidade , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Adiposidade/efeitos dos fármacos , Animais , Compostos Benzidrílicos/toxicidade , Diferenciação Celular , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , PPAR gama/genética , Fenóis/toxicidade , GravidezRESUMO
INTRODUCTION: Brain iron homeostasis dysregulation has been widely related to neurodegeneration. In particular, human haemochromatosis protein (HFE) is involved in iron metabolism, and HFE H63D polymorphism has been related to the risk of amyotrophic lateral sclerosis and Alzheimer's disease. Recently, iron accumulation in the basal ganglia of frontotemporal lobar degeneration (FTLD) patients has been described. OBJECTIVE: To explore the relationship between HFE genetic variation and demographic, clinical and imaging characteristics in a large cohort of FTLD patients. METHODS: A total of 110 FTLD patients underwent neuropsychological and imaging evaluation and blood sampling for HFE polymorphism determination. HFE H63D polymorphism was considered in the present study. Two imaging approaches were applied to evaluate the effect of HFE genetic variation on brain atrophy, namely voxel-based morphometry and region of interest-based probabilistic approach (SPM8; Wellcome Trust Centre for Neuroimaging). RESULTS: FTLD patients carrying the D* genotype (H/D or D/D) showed greater atrophy in the basal ganglia, bilaterally, compared to H/H carriers (x, y, z: -22, -4, 0; T = 3.45; cluster size: 33 voxels, x, y, z: 24, 4, -2; T = 3.38; cluster size: 36 voxels). The former group had even more pronounced behavioural symptoms, as defined by the Frontal Behavioural Inventory total scores. CONCLUSIONS: Our data suggest that H63D polymorphism could represent a disease-modifying gene in FTLD, fostering iron deposition in the basal ganglia. This suggests a new possible mechanism of FTLD-associated neurodegeneration.
Assuntos
Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Degeneração Lobar Frontotemporal/diagnóstico por imagem , Degeneração Lobar Frontotemporal/metabolismo , Proteína da Hemocromatose/genética , Ferro/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Atrofia , Encéfalo/patologia , Estudos de Coortes , Feminino , Degeneração Lobar Frontotemporal/genética , Degeneração Lobar Frontotemporal/psicologia , Frequência do Gene , Substância Cinzenta/diagnóstico por imagem , Substância Cinzenta/metabolismo , Substância Cinzenta/patologia , Humanos , Imageamento por Ressonância Magnética , Masculino , Testes Neuropsicológicos , Tamanho do Órgão , Projetos Piloto , Polimorfismo GenéticoRESUMO
PURPOSE: Several nutrients act as phytoestrogens, being anti-adipogenic when consumed with a fat-rich diet. Their effect on a low-fat diet (LFD) background is unknown. We tested soy and genistein effects on adipose tissue in LFD-fed mice and genistein activity in the 3T3-L1 adipogenesis model. METHODS: C57BL/6 J male mice were fed an 8.5% soy-supplemented LFD (SS-LFD) or a soy-free LFD (SF-LFD) for 147 days. Groups of 3-week-old (pubertal) and 6-week-old (adult) mice on the SF-LFD were also treated with 17ß-estradiol (E2, 5 µg/kg/day) ip or pure genistein (5 mg/kg/day) by gavage for 15 days. Body fat deposition and gene expression profiles were evaluated. E2 and genistein effects on ERα, ERß and PPARγ transcriptional activities were characterized in ERα- or ERß-transfected 3T3L1 cells during differentiation, by the use of reporter plasmids. RESULTS: The SS-LFD group increased fat mass compared with the SF-LFD group. Genistein alone increased while E2 decreased fat pads in the 15-day-treated mice. In visceral fat, genistein differentially regulated 13 metabolic pathways compared to E2. PPARγ-controlled genes were downregulated by E2, while they were upregulated by genistein. In 3T3-L1 cells, genistein activated ERß-driven transcription, differentiation and lipid accumulation, while inhibited ERα-driven transcription, without effects on lipid accumulation. E2 activated both ERs only in preadipocytes. In differentiated untransfected cells, genistein inhibited PPARγ, while activated PPARγ in the presence of ERß. CONCLUSIONS: Soy and genistein at nutritional doses induce fat development in LFD-fed mice and adipogenesis in 3T3-L1 cells, with a mechanism that involves, at least in vitro, ERß and is dependent on cell differentiation stage.
Assuntos
Tecido Adiposo/efeitos dos fármacos , Dieta com Restrição de Gorduras , Genisteína/administração & dosagem , Glycine max/química , Células 3T3-L1 , Adipogenia/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Regulação para Baixo , Estradiol/administração & dosagem , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Genisteína/sangue , Teste de Tolerância a Glucose , Masculino , Camundongos , Camundongos Endogâmicos C57BL , PPAR gama/genética , PPAR gama/metabolismo , TranscriptomaRESUMO
Cell differentiation and response to hormonal signals were studied in a 3D environment on an in-house generated mouse fibroblast cell line expressing a reporter gene under the control of estrogen responsive sequences (EREs). 3D cell culture conditions were obtained in a Rotary Cell Culture System; (RCCS™), a microgravity based bioreactor that promotes the aggregation of cells into multicellular spheroids (MCS). In this bioreactor the cells maintained a better differentiated phenotype and more closely resembled in vivo tissue. The RCCS™ cultured fibroblasts showed higher expression of genes regulating cell assembly, differentiation and hormonal functions. Microarray analysis showed that genes related to cell cycle, proliferation, cytoskeleton, migration, adhesion and motility were all down-regulated in 3D as compared to 2D conditions, as well as oncogene expression and inflammatory cytokines. Controlled remodeling of ECM, which is an essential aspect of cell organization, homeostasis and tissue was affected by the culture method as assessed by immunolocalization of ß-tubulin. Markers of cell organization, homeostasis and tissue repair, metalloproteinase 2 (MMP2) and its physiological inhibitor (TIMP4) changed expression in association with the relative formation of cell aggregates. The fibroblasts cultured in the RCCS™ maintain a better responsiveness to estrogens, measured as expression of ERα and regulation of an ERE-dependent reporter and of the endogenous target genes CBP, Rarb, MMP1 and Dbp. Our data highlight the interest of this 3D culture model for its potential application in the field of cell response to hormonal signals and the pharmaco-toxicological analyses of chemicals and natural molecules endowed of estrogenic potential.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Estrogênios/fisiologia , Matriz Extracelular/fisiologia , Fibroblastos/fisiologia , Animais , Perfilação da Expressão Gênica , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE AND BACKGROUND: The focus was directed to the study of two of the most lignan-rich food sources: sesame and flaxseeds. Recent epidemiological and experimental evidences suggesting that these foods may improve metabolic functions underlying metabolic syndrome (MetS). METHODS: To characterize the effect of these oilseeds on metabolic functions, we conducted an experimental study aimed at preventing adiposity and metabolic imbalance in a mouse model of high-fat diet (HFD)-induced MetS. Statistical analysis was performed by two-way analysis of variance test followed by post hoc Bonferroni analysis. RESULTS: We studied the effect of the oilseeds sesame and flaxseed on metabolic parameters in mice on a HFD. When the HFD was integrated with 20% of sesame or flaxseed flours, the mice showed a decrease in body fat, already at day 15, from time 0. The size of the adipocytes was smaller in epididymal fat, liver steatosis was inhibited, and insulin sensitivity was higher in mice on the supplemented diets. The supplemented diets also resulted in a significant increase in the serum levels of the lignan metabolites enterodiol and enterolactone compared with the controls. The expression of genes associated with the inflammatory response, glucose metabolism, adipose metabolism and nuclear receptor were altered by the oilseed-supplemented diets. Some of the most abundant lignans in these oilseeds were studied in 3T3-L1 preadipocyte cells and were effective in inhibiting adipocyte differentiation at the minimal dose of 1 nM. CONCLUSIONS: The consumption of sesame and flaxseed may be beneficial to decrease metabolic parameters that are generally altered in MetS.
Assuntos
Adipócitos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Óleo de Semente do Linho/farmacologia , Óleo de Gergelim/farmacologia , Células 3T3-L1 , 4-Butirolactona/análogos & derivados , 4-Butirolactona/sangue , Adipócitos/metabolismo , Tecido Adiposo/citologia , Adiposidade , Animais , Dieta Hiperlipídica , Gorduras na Dieta/administração & dosagem , Modelos Animais de Doenças , Resistência à Insulina , Lignanas/sangue , Masculino , Síndrome Metabólica/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Anemia is the main extraintestinal comorbidity of Inflammatory Bowel Disease (IBD). Differentiating the type of anemia in these disorders is still a challenge. Hepcidin could be a promising biomarker to identify iron deficiency anemia (IDA), anemia of chronic disease (ACD) and the concomitant presence of both IDA and ACD. METHODS: To evaluate the potential role of hepcidin dosage in the management of anemia in IBD patients, we performed a systematic review by a comprehensive literature analysis of original papers reporting the dosage of hepcidin in IBD patients. In all the articles reviewed, the dosage of ferritin was reported, and the correlation between hepcidin and ferritin has been used to compare these two biomarkers. RESULTS: A total of 12 articles concerning the dosage of hepcidin in IBD were included, comprising in total of 976 patients. The results of the hepcidin values in IBD patients when compared with controls were conflicting. In fact, four articles described an increase in this biomarker, three showed a decrease and five did not find significant differences. The correlation with ferritin was positive and significant. In three studies, some differences between hepcidin dosages and ferritin levels indicate a possible role when IDA and ACD could be present at the same time. CONCLUSIONS: Considering the contradictory data of the studies, the diagnostic role of hepcidin as a biomarker remains elusive in IBD patients. These differences could be due to the clinical characteristics of the patients enrolled that should be better defined in the future. A suitable clinical trial should be designed to outline the possible role of hepcidin in differentiating IDA, ACD and concomitant IDA and ACD in IBD patients. At the moment, ferritin still remains the best marker to diagnose these conditions, in addition to hemoglobin, transferrin saturation and CRP as recommended by the ECCO guidelines.