RESUMO
Cerebrovascular events (CE) are one of the most common and severe events in antiphospholipid syndrome (APS), a condition characterized by thrombosis and circulating anti-phospholipid antibodies (aPL). Seronegative APS (SN-APS) refers to a group of patients with clinical features of APS but persistently negative tests for "criteria aPL": anti-cardiolipin antibodies (aCL) and anti-ß2glycoprotein I antibodies detected by enzyme-linked immunosorbent assay (ELISA), and the lupus anticoagulant detected by clotting assays. We report a series of five cases of SN-APS in young or middle-aged patients who tested positive for "non-criteria" aPL. We retrospectively collected cases of SN-APS patients who experienced CE without an identified cause despite an extensive diagnostic work-up and tested negative for criteria aPL. All the patient sera were tested for aCL by immunostaining on thin-layer chromatography (TLC) and anti-vimentin/cardiolipin (aCL/Vim) by ELISA. We identified five cases of female patients aged 21 to 58 years, evaluated at the Rheumatology Unit and/or Stroke Unit/Emergency Department of the Sapienza University Hospital of Rome, "Policlinico Umberto I". All patients presented a clinical history suggestive of APS. All the patients tested positive for aCL by TLC-immunostaining, and one patient was positive for aCL/Vim. In young or middle-aged patients with cryptogenic CE and a clinical history suggestive of APS, the use of new diagnostic tools for identifying aPL, if validated in future studies, could represent an important step in the prompt diagnosis of APS.
RESUMO
BACKGROUND: Autophagy has emerged as a key mechanism in the survival and function of T and B lymphocytes, and its activation was involved in apoptosis resistance in rheumatoid arthritis (RA). To investigate whether the relationship between autophagy and apoptosis may impact the response to the therapy, we analyzed ex vivo spontaneous autophagy and apoptosis in patients with RA subjected to treatment with anti-tumor necrosis factor (TNF) drugs and in vitro the effects of TNFα and anti-TNF drugs on cell fate. METHODS: Peripheral blood mononuclear cells (PBMCs) from 25 RA patients treated with anti-TNF drugs were analyzed for levels of autophagy marker LC3-II by western blot and for the percentage of annexin V-positive apoptotic cells by flow cytometry. The same techniques were used to assess autophagy and apoptosis after in vitro treatment with TNFα and etanercept in both PBMCs and fibroblast-like synoviocytes (FLS) from patients with RA. RESULTS: PBMCs from patients with RA responsive to treatment showed a significant reduction in LC3-II levels, associated with an increased apoptotic activation after 4 months of therapy with anti-TNF drugs. Additionally, the expression of LC3-II correlated with DAS28. TNFα was able to induce autophagy in a dose-dependent manner after 24 h of culture in RA PBMCs and FLS. Moreover, etanercept caused a significant reduction of autophagy and of levels of citrullinated proteins. CONCLUSIONS: Our results show how the crosstalk between autophagy and apoptosis can sustain the survival of immune cells, thus influencing RA progression. This suggests that inhibition of autophagy represents a possible therapeutic target in RA.
Assuntos
Apoptose/efeitos dos fármacos , Artrite Reumatoide/tratamento farmacológico , Autofagia/efeitos dos fármacos , Etanercepte/uso terapêutico , Metotrexato/uso terapêutico , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Idoso , Antirreumáticos/uso terapêutico , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Células Cultivadas , Etanercepte/metabolismo , Feminino , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de Saúde , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Anti-phospholipid syndrome (APS) is characterized by arterial and/or venous thrombosis and pregnancy morbidity. It is well known that in these patients thrombosis may be the result of a hypercoagulable state related to anti-ß2-glycoprotein I (ß2-GPI) antibodies. Moreover, platelets may play a role in thrombotic manifestations by binding of anti-ß2-GPI antibodies. Platelets express tissue factor (TF), the major initiator of the clotting cascade, after activation. We primarily analyzed whether anti-ß2-GPI antibodies may trigger a signal transduction pathway leading to TF expression in human platelets. Platelets from healthy donors were incubated with affinity purified anti-ß2-GPI antibodies for different times. Platelet lysates were analyzed for phospho-interleukin-1 receptor-associated kinase 1 (IRAK), phospho-p65 nuclear factor kappaB (NF-κB) and TF by Western blot. IRAK phosphorylation was observed as early as 10 min of anti-ß2-GPI treatment, with consequent NF-κB activation, whereas TF expression, detectable at 45 min, was significantly increased after 4 h of anti-ß2-GPI treatment. Virtually no activation was observed following treatment with control immunoglobulin IgG. We then analyzed TF expression in platelets from 20 APS patients and 20 healthy donors. We observed a significant increase of TF in APS patients versus control subjects (P < 0·0001). This work demonstrates that anti-ß2-GPI antibodies may trigger in vitro a signal transduction pathway in human platelets, which involves IRAK phosphorylation and NF-κB activation, followed by TF expression. Furthermore, ex vivo, platelets of APS patients showed a significantly increased expression of TF. These findings support the view that platelets may play a role in the pathogenesis of APS, with consequent release of different procoagulant mediators, including TF.
Assuntos
Síndrome Antifosfolipídica/imunologia , Plaquetas/fisiologia , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Tromboplastina/metabolismo , beta 2-Glicoproteína I/imunologia , Adulto , Formação de Anticorpos , Autoanticorpos/metabolismo , Coagulação Sanguínea , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Fosforilação , Transdução de Sinais , Tromboplastina/genética , Transgenes/genéticaRESUMO
It was shown that receptor-mediated apoptosis involves a cascade of subcellular events including alterations of mitochondria. Loss of mitochondrial membrane potential that follows death receptor ligation allows the release of apoptogenic factors that result in apoptosis execution. Further important mitochondrial changes have been observed in this regard: mitochondrial remodeling and fission that appear as prerequisites for the occurrence of the cell death program. As it was observed that lipid rafts, glycosphingolipid-enriched structures, can participate in the apoptotic cascade being recruited to the mitochondria under receptor-mediated proapoptotic stimulation, we decided to analyze the possible implication of these microdomains in mitochondrial fission. We found that molecules involved in mitochondrial fission processes are associated with these domains. In particular, although hFis1 was constitutively included in mitochondrial raft-like domains, dynamin-like protein 1 was recruited to these domains on CD95/Fas triggering. Accordingly, the disruption of rafts, for example, by inhibiting ceramide synthase, leads to the impairment of fission molecule recruitment to the mitochondria, reduction of mitochondrial fission and a significant reduction of apoptosis. We hypothesize that under apoptotic stimulation the recruitment of fission-associated molecules to the mitochondrial rafts could have a role in the morphogenetic changes leading to organelle fission.
Assuntos
Apoptose , Microdomínios da Membrana/química , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/química , Proteínas Mitocondriais/análise , Células Cultivadas , Centrifugação com Gradiente de Concentração , Dinaminas , Inibidores Enzimáticos/farmacologia , Fumonisinas/farmacologia , GTP Fosfo-Hidrolases/análise , Gangliosídeos/análise , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/análise , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Octoxinol , Oxirredutases/antagonistas & inibidores , Interferência de RNA , Receptor fas/metabolismoRESUMO
Plasma membrane lipid microdomains have been considered as a sort of 'closed chamber', where several subcellular activities, including CD95/Fas-mediated proapoptotic signaling, take place. In this work we detected GD3 and GM3 gangliosides in isolated mitochondria from lymphoblastoid CEM cells. Moreover, we demonstrated the presence of microdomains in mitochondria by immunogold transmission electron microscopy. We also showed that GD3, the voltage-dependent anion channel-1 (VDAC-1) and the fission protein hFis1 are structural components of a multimolecular signaling complex, in which Bcl-2 family proteins (t-Bid and Bax) are recruited. The disruption of lipid microdomains in isolated mitochondria by methyl-beta-cyclodextrin prevented mitochondria depolarization induced by GD3 or t-Bid. Thus, mitochondrion appears as a subcompartmentalized organelle, in which microdomains may act as controllers of their apoptogenic programs, including fission-associated morphogenetic changes, megapore formation and function. These results disclose a new scenario in which mitochondria-associated lipid microdomains can act as regulators and catalysts of cell fate.
Assuntos
Apoptose/fisiologia , Gangliosídeo G(M3)/metabolismo , Microdomínios da Membrana/metabolismo , Mitocôndrias/metabolismo , Linfócitos T/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Proteínas de Membrana , Microscopia Confocal , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Linfócitos T/citologia , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Proteína X Associada a bcl-2/metabolismo , beta-Ciclodextrinas/farmacologia , Receptor fas/metabolismoRESUMO
It has been shown that human calcitonin (hCT) is absorbed through the nasal mucosa when administered together with promoters like sodium glycocholate (SGC) or dihydrofusinate. The aim of this study was to compare the clinical and metabolic effect of intranasal (in) and intramuscular (im) hCT in patients with osteoporosis or with Paget's disease of bones. Fifteen women with postmenopausal or with senile osteoporosis entered a randomized six months trial with in hCT (plus SGC) or with im hCT 100 U on alternate days. Six women in each group were treated for 2 months, and only four women in each group continued treatment for an additional 4 months period. In hCT, but not im hCT, reduced subjective pain, while urinary cAMP increased to a similar extent in the 2 groups. Other metabolic indexes and bone mineral content (BMC) were unchanged, no new fractures took place, and side effects were fewer with in than with im hCT. To confirm the analgesic effect of in hCT, twelve patients with Paget's disease of bone were randomly treated for 20 days with in or im hCT 100 U/day: during the short period of treatment, pain was reduced by in, not by im hCT, and urinary cAMP excretion similarly increased in the two groups of patients.
Assuntos
Calcitonina/uso terapêutico , Osteíte Deformante/tratamento farmacológico , Osteoporose/tratamento farmacológico , Administração Intranasal , Idoso , Densidade Óssea/efeitos dos fármacos , Calcitonina/administração & dosagem , Calcitonina/farmacologia , AMP Cíclico/urina , Feminino , Humanos , Injeções Intramusculares , Pessoa de Meia-Idade , Osteíte Deformante/metabolismo , Osteoporose/metabolismo , Osteoporose Pós-Menopausa/tratamento farmacológico , Osteoporose Pós-Menopausa/metabolismo , Dor/tratamento farmacológico , Projetos PilotoRESUMO
Cefoperazone is a third-generation semisynthetic injectable cephalosporin. It has been reported that cefoperazone has beta-lactamase resistance and quite a broad antimicrobial spectrum against many Gram-positive and Gram-negative microorganisms and most anaerobes. In this study, the pharmacokinetics of cefoperazone were examined in a group of 10 patients suffering from acute exacerbations of chronic bronchitis, with purulent or mucopurulent expectorations. Cefoperazone was administered at the dose of 1 g i.m. every 12 hours. Serum and urinary parameters and the profile of bronchial mucus diffusion were assessed after the first administration and during the whole period of treatment which lasted for 7 days. In a second, third, and fourth group of volunteer patients who had to undergo surgical operations, bone, lung and prostatic penetration of cefoperazone was determined in correlation with serum levels.
Assuntos
Osso e Ossos/metabolismo , Bronquite/metabolismo , Cefoperazona/metabolismo , Pulmão/metabolismo , Próstata/metabolismo , Escarro/análise , Bronquite/tratamento farmacológico , Humanos , Injeções Intramusculares , Cinética , MasculinoAssuntos
Bronquite/metabolismo , Leucomicinas/metabolismo , Humanos , Cinética , Distribuição TecidualRESUMO
Cefotaxime is a new powerful methoxycephalosporin with a broad anti-microbial spectrum, suitable for parenteral administration. In the present study, the concentrations of cefotaxime in serum and in bronchial secretion were determined after intramuscular injection of 1 gm every eight hours for seven days. Subjects were patients suffering from an exacerbation of chronic bronchitis. Serum levels versus time curve were interpreted in terms of a one-compartment open model. Pharmacokinetic parameters after single and multiple doses were investigated. No evidence of significant accumulation was found. Furthermore, a type of in vivo rate of killing with cefotaxime was investigated by evaluating the decrease in the number of colonies in bronchial mucus cultures daily for seven days. In two groups of volunteers who had to undergo surgery, bone and prostatic concentrations of cefotaxime were determined and correlated with serum levels.