RESUMO
Artichoke is a characteristic crop of the Mediterranean area, recognized for its nutritional value and therapeutic properties due to the presence of bioactive components such as polyphenols, inulin, vitamins and minerals. Artichoke is mainly consumed after home and/or industrial processing, and the undersized heads, not suitable for the market, can be used for the recovery of bioactive compounds, such as polyphenols, for cosmetic applications. In this paper, the potential skin anti-age effect of a polyphenolic artichoke extract on endothelial cells was investigated. The methodology used was addressed to evaluate the antioxidant and anti-inflammatory activities and the improvement of gene expression of some youth markers. The results showed that the artichoke extract was constituted by 87% of chlorogenic, 3,5-O-dicaffeoylquinic, and 1,5-O-dicaffeoylquinic acids. The extract induced important molecular markers responsible for the microcirculation and vasodilatation of endothelial cells, acted as a potential anti-inflammatory agent, protected the lymphatic vessels from oxidative damage by ROS formation, and enhanced the cellular cohesion by reinforcing the tight junction complex. In addition, the artichoke extract, through the modulation of molecular pathways, improved the expression of genes involved in anti-ageing mechanisms. Finally, clinical testing on human subjects highlighted the enhancement by 19.74% of roughness and 11.45% of elasticity from using an artichoke extract cosmetic formulation compared to placebo cream.
Assuntos
Cynara scolymus/química , Células Endoteliais/efeitos dos fármacos , Extratos Vegetais/farmacologia , Polifenóis/farmacologia , Envelhecimento da Pele/efeitos dos fármacos , Biomarcadores , Cromatografia Líquida de Alta Pressão , Células Endoteliais/metabolismo , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Macrófagos , Óxido Nítrico/metabolismo , Compostos Fitoquímicos/química , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/química , Polifenóis/química , Adulto JovemRESUMO
BACKGROUND: Proteasomes are 'proteolytic machineries' implicated in many cellular functions, including protein turnover, inflammatory response and immunosurveillance. They exist in various forms sharing the same catalytic core - the 20S proteasome. This core consists of 28 subunits codified by 14 different genes, 3 of which - beta 1, beta 2 and beta 5 - are catalytically active and show peptidyl-glutamyl peptide hydrolyzing (PGPH), trypsin-like and chymo-trypsin-like activities, respectively. Under IFN- delta and TNF- alfa stimuli, the 3 active constitutive subunits are replaced by the corresponding ones - i.e., LMP2, MECL-1, LMP7 - known as inducible subunits, thus resulting in the constitution of the 'immunoproteasome' that is specifically implicated in MHC class I-presented peptide generation. This process is enhanced when the proteasome is associated with the polymeric protein 11S regulator/PA28 made up of 4 alfa and 3 beta subunits. METHODS: The 20S proteasome was purified from post mortem specimens of human kidney cortex by chromatographic and ultracentrifugation techniques. It was then characterized on the basis of (i) multicatalytic activity evaluated using specific fluorogenic peptides, (ii) electrophoretic mobility on non-denaturating polyacrylamide gels followed by in-gel visualization by fluorogenic peptide overlaying and Coomassie blue staining and (iii) subunit composition as ascertained by SDS-PAGE and 2-dimensional electrophoresis followed by silver staining or Western immunoblotting using specific antibodies against the proteasome subunits. The 20S proteasome was also studied for its association with the 11S regulator by Western immunoblotting using an antibody to the regulator alfa subuniT. RESULTS: T he purified proteasome was shown to have PGPH, trypsin-like and chymotrypsin-like activities. Furthermore, it incorporated the inducible subunits and was associated with the 11S regulator. CONCLUSIONS: The features we observed make renal cells susceptible to an over-expression of inflammatory response to immunological challenges.
Assuntos
Córtex Renal/enzimologia , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/isolamento & purificação , Humanos , Inflamação/enzimologia , Inflamação/genética , Inflamação/imunologia , Córtex Renal/imunologia , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/imunologia , Estrutura Quaternária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato/fisiologiaRESUMO
Immune and inflammatory response activation is a common feature of connective tissue diseases and systemic vasculitis. The aim of our study was to evaluate the possible involvement of TNFalpha c.-308A > G, IL-10 c.-1082A > G, uteroglobin c.38A > G, TGFbeta 1 c.869C > T and NFkappaB2 c.-1837T > C gene polymorphisms in susceptibility to connective tissue diseases. Our study cohort included 68 unrelated patients affected by rheumatoid arthritis (RA) (37 patients) and ANCA-positive [micropolyangiitis (mPA) 17 patients] or ANCA-negative systemic vasculitis [including 8 patients with Henoch-Schönlein purpura (HSP) and 6 patients with mixed cryoglobulinaemia (MC)] as well as 98 control subjects. Allele frequency analysis of uteroglobin c.38G > A polymorphism showed a significant increase in the c.38A allele in patients (p= 0.002). Genotype frequency analysis of uteroglobin and NF-kappaB2 gene polymorphisms in patients showed an increase in c.38GA and c.38AA genotypes in the uteroglobin gene (p=0.02) coupled with an increase in homozygous c.-1837CC in the NF-kappaB2 gene (p=0.02). Our data suggest that genetic variation in UG and NF-kappaB2 pathways could have effects in connective tissue disease susceptibility.
Assuntos
Artrite Reumatoide/genética , Polimorfismo Genético , Vasculite Sistêmica/genética , Sequência de Bases , Citocinas/genética , Primers do DNA , Feminino , Predisposição Genética para Doença , Humanos , MasculinoRESUMO
Type II mixed cryoglobulinemia (MC) is a systemic vasculitis, associated in most cases with hepatitis C virus (HCV) infection, sustained by proliferation of oligoclonal cells. Systemic B cell depletion and clinical remission can be achieved in non-Hodgkin lymphoma by human/mouse chimeric monoclonal antibody that specifically reacts with the CD20 antigen (rituximab). Similar effects could be expected in type II MC. Twelve patients, mean age 61.9 years (range 37-76), 11 with HCV infection genotype 2a2c (4 cases) or 1b (6 cases) and 3 (1 case) and symptomatic type II MC with systemic manifestations, including renal involvement, marrow clonal restriction, large necrotizing ulcers, and polyneuropathy, were considered eligible for rituximab therapy because of resistance or intolerance to conventional therapy or important bone marrow infiltration. Rituximab was administered intravenously at a dose of 375 mg/m2 on days 1, 8, 15, and 22. Two more doses were administered 1 and 2 months later. No other immunosuppressive drugs were added. Response was evaluated by assessing the changes in clinical signs, symptoms, and laboratory parameters. Levels of proteinuria, hematuria, erythrocyte sedimentation rate, cryocrit, rheumatoid factor, and IgM decreased while C4 values increased and HCV viral load remained stable during short- and medium-term observation. Bone marrow abnormalities were found to reverse to normal. Constitutional symptoms disappeared or ameliorated. No acute or delayed side effects were seen. Based on this experience and a number of reports published in the last 5 years, Rituximab appears to be a safe and effective therapeutic option in symptomatic patients with HCV-associated MC with signs of systemic vasculitis.
Assuntos
Anticorpos Monoclonais , Crioglobulinemia/tratamento farmacológico , Glomerulonefrite/tratamento farmacológico , Hepatite C/complicações , Fatores Imunológicos , Adulto , Idoso , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Murinos , Ensaios Clínicos como Assunto , Crioglobulinemia/imunologia , Crioglobulinemia/fisiopatologia , Glomerulonefrite/imunologia , Glomerulonefrite/fisiopatologia , Hepatite C/tratamento farmacológico , Humanos , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/uso terapêutico , Pessoa de Meia-Idade , Rituximab , Índice de Gravidade de Doença , Resultado do TratamentoRESUMO
AIM: The aim of the study was to evaluate biocompatibility of anew polystyrene-type adsorbent (BetaSorb) designed for hemoperfusion, using second-level biomolecular analyses. The device has recently been developed to enhance beta2-microglobulin removal during hemodialysis. Molecular structure and chemical modifications of the surface beads of this cartridge should prevent exposure of dense hydrophobic surface sites to proteins, and avoid the major drawbacks of previous polystyrene-type adsorbent materials. METHODS: Whole blood of healthy donors was incubated in sterile minicolumns packed with BetaSorb Cuprophan, Hemophan, polysulfone and cellulose acetate. In parallel experiments, whole blood was recirculated for 180 min in a sham dialysis circuit equipped with the study sorbent or Hemophan or polysulfone. Biocompatibility was assessed by means of new biomolecular approaches focused on nuclear factor kappaB (NF-kappaB) activation (assessed by electrophoretic mobility shift assay), TNF-alpha and IL-1beta gene expression (evaluated by real-time PCR), TNF-alpha and IL-1beta production (measured by Western blot assay and ELISA), nitric oxide (NO) generation (detected by electron paramagnetic resonance), free oxygen radical production (by chemiluminescence in a biological assay) and the generation of the complement breakdown product C3d. RESULTS: In coincubation experiments, 5-min contact with any dialysis device, but BetaSorb, was enough to induce activation of NF-kappaB. The amount of TNF-alpha precursor form was found to increase after 5 min of exposure to each tested polymer, but no traces of mature forms of TNF-alpha or IL-1beta were detected in in vitro experimental conditions using healthy blood. NO and free oxygen radical generation were significantly lower in blood samples exposed to BetaSorb than in control dialysis devices. C3d levels were found to be increased with Hemophan, unaffected by polysulfone, and remarkably decreased with the BetaSorb device. In the sham hemodialysis experiments, NF-kappaB activation and C3d and NO profiles were similar to direct incubation experiments. Compared to basal levels, quantitation of TNF-alpha and IL-1beta mRNA revealed a 15- and 9-fold increase, respectively, in samples exposed to Hemophan for 180 min. CONCLUSIONS: The new BetaSorb device not only appears to be highly biocompatible, but shares properties that make it probably able to interfere with the activation of the inflammatory state.
Assuntos
Hemoperfusão/métodos , NF-kappa B/metabolismo , Poliestirenos/química , Adsorção , Citocinas/metabolismo , Expressão Gênica , Hemoglobinas/análise , Hemoperfusão/instrumentação , Humanos , Interleucina-1/análise , Interleucina-1/genética , Interleucina-1/metabolismo , Medições Luminescentes/métodos , Teste de Materiais/métodos , NF-kappa B/sangue , Óxido Nítrico/análise , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Among the possible variables associated with cytokine activation in hemodialysis, filter membrane has been reported to be a major factor and monocytes adherent to the membrane have been suggested as a possible source for cytokine synthesis. METHODS: In order to exclude variables other than the direct cell-to-membrane interaction, suspensions of peripheral blood monocytes isolated from donors' blood were incubated for 180 min at 37 degrees C in Petri dishes coated with cuprophan (Cu) or polyacrylonitrile (AN69S) or polysulfone (PS). Total RNA was purified, reverse transcribed in cDNA and amplified by polymerase chain reaction (PCR) primed with specific oligomers for determining IL-1beta and TNF-alpha gene expression. For Western blot analysis, cell homogenates and supernatants were electrophoresed and transferred to a polyvinylidene difluoride membrane and the membrane was then incubated with polyclonal antibodies specific for the detection of IL-1beta and TNF-alpha. RESULTS: Semi-quantitation of targets for PCR (using the technique of limiting dilutions and referring to actin and glyceraldehyde-3-phosphate dehydrogenase as noninducible molecule) revealed a comparable increase at 180 min (p < 0.05) in IL-1beta and TNF-alpha mRNA in monocytes incubated with polyacrylonitrile, PS and Cu membranes. In 2 of 10 experiments pro-IL-1beta was detected in monocytes interacting with Cu, PS and AN69S membrane. However, the extent of extracellular release of mature protein was greater for Cu. CONCLUSION: Some dissociation between transcriptional and translational events was detected in experiments using donors' blood in vitro. More specifically, synthetic membranes (both polyacrylonitrile and PS) were found to be as active as Cu in inducing IL-1beta and TNF-alpha mRNA expression, but less effective in promoting the extracellular release of proinflammatory cytokine products.
Assuntos
Interleucina-1/biossíntese , Membranas Artificiais , Monócitos/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Expressão Gênica , Humanos , Técnicas In Vitro , Interleucina-1/genética , Rins Artificiais , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Fator de Necrose Tumoral alfa/genéticaRESUMO
Uteroglobin (UG) is a multifunctional protein with anti-inflammatory/immunomodulatory properties. The UG gene is located on the long arm of chromosome 11 (11q12.3-q13.1) in a region linked to some immune disorders. A guanine-adenine substitution at position 38 (A38G) has been found in the noncoding region of exon 1 that is significantly correlated with an increased risk of developing immune-mediated diseases. Recently an experimental model of UG knockout mice showed that in mice, UG deficiency causes severe glomerulopathy with mesangial deposition of IgA-fibronectin complexes. To detect the presence of polymorphisms in the UG coding sequence, the DNA of 109 patients with IgA nephropathy (IgAN), and 32 patients with systemic lupus erythematosus (SLE) were tested for the nucleotide sequence of all three UG exons by heteroduplex analysis. We detected heterozygous DNA only for exon 1 due to the A38G substitution, as confirmed by sequencing. We tested for A38G polymorphism, by restriction endonuclease digestion (Sau96I), both in SLE patients and in IgAN patients. Twenty patients with either membranous nephropathy (12) or focal and segmental glomerular sclerosis and 120 healthy subjects served as controls. Compared with both healthy controls and non-IgA control patients, the frequency of the 38A allele was significantly higher in SLE patients (38 of 64 alleles versus 89 of 240 alleles, p = 0.002, and versus 7 of 40 alleles, p < 0.001). IgAN patients showed an allelic distribution similar to both control groups. A subgroup of 18 IgAN patients undergoing renal replacement therapy because of end-stage renal disease showed a significant increase in 38A allele frequency (5 of 36 38G alleles versus 31 of 36 38A alleles, p < 0.001). UG is an immunomodulatory agent that is able to (a) inhibit the activity of several phospholipase A2 (PLA2s), (b) interfere with the function of both neutrophils and monocytes, and (c) prevent immune recognition, perhaps by masking surface antigens. This could account for the role this molecule plays in SLE. The A38G polymorphism is located within a region corresponding to the rat minimal promoter that proved to be important in the transcriptional regulation of UG. Although the significance of any alterations in the UG exon 1 noncoding region in humans has yet to be clarified, initial evidence suggests that it may alter the control of immune response and of inflammation.