RESUMO
Diffuse large B-cell lymphoma (DLBCL) is the most common and one of the most aggressive subtypes of non-Hodgkin's lymphomas. Front-line therapy consists of chemotherapy in combination with anti-CD20 monoclonal antibody rituximab. Relapses after rituximab-based regimen have poor prognosis and call for new treatment options. Immunohistochemistry analysis of relapsed DLBCL often reveal CD20-negative lymphoma, which limits repeated use of rituximab in combination with salvage chemotherapy. CD38 is a surface antigen that binds to CD38, CD31/PECAM-1 and hyaluronic acid. CD38 is an important mediator of signal transmission from the microenvironment into the cell. Anti-CD38 monoclonal antibody daratumumab has been approved for the treatment of multiple myeloma. Expression of CD38 on the surface of DLBCL is highly variable (compared to strong expression on myeloma cells), but can be easily assessed by flow cytometry or immunohistochemistry. A patient-derived xenograft (PDX) model of CD20-negative, CD38-positive DLBCL derived from a patient with rituximab-refractory DLBCL was used for in vivo experiments. We demonstrated that daratumumab suppressed growth of subcutaneous PDX tumours significantly more effectively than rituximab. Analysis of tumours obtained from mice treated with daratumumab revealed down-regulation of surface CD38, suggesting endocytosis of CD38-daratumumab complexes. The results suggest a potential clinical use of daratumumab in combination with salvage chemotherapy in patients with relapses of CD20-negative DLBCL. In addition, daratumumab might potentially serve as a suitable antibody moiety for derivation of antibodydrug conjugates for the targeted delivery of toxic payloads to the lymphoma cells.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos , Linfoma Difuso de Grandes Células B , ADP-Ribosil Ciclase 1/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Glicoproteínas de Membrana/antagonistas & inibidores , Camundongos , Rituximab/uso terapêutico , Microambiente TumoralRESUMO
Iron overload causes tissue damage in the liver, but its initial effects at the molecular and cellular level are not well understood. Epithelial cadherin (E-cad) is a major adhesion protein in adherens junctions and is associated with several signal transduction pathways. Dysfunction of E-cad causes instability of adherens junctions, which leads to cell invasion, cell migration, and carcinogenesis. We found in liver samples from iron-overloaded mice that the apparent molecular mass of E-cad was reduced from 125 to 115 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing conditions and immunoblotting, and that the cellular expression of E-cad was decreased in immunohistochemistry. The mRNA level of E-cad, however, did not change significantly, suggesting that the alterations are posttranslational. Interestingly, incubation of control liver extracts with Fe2+ alone also produced the same mobility shift. Neither an oxidant nor an antioxidant influenced this shift in vitro, suggesting that reactive oxygen species, which are generated by iron and known to cause damage to macromolecules, are not involved. Treatment of the 115 kDa E-cad with deferoxamine, an iron chelator, thus removing Fe2+, shifted the molecular mass back to 125 kDa, demonstrating that the shift is reversible. The observation also implies that the alteration that causes the mobility shift is not due to transcriptional control, deglycosylation, and proteolysis. This reversible mobility shift of E-cad has not been previously known. The alteration of E-cad that causes the mobility shift might be an initial step to liver diseases by iron overload.
Assuntos
Caderinas/química , Sobrecarga de Ferro/fisiopatologia , Fígado/fisiopatologia , Animais , Ensaio de Desvio de Mobilidade Eletroforética , Fígado/química , Camundongos , Processamento de Proteína Pós-TraducionalRESUMO
Cancer is known to be an important cause of anaemia due to several factors including iron deficiency and inflammation. Hepcidin, a key regulator of iron metabolism, is up-regulated by iron and inflammatory stimuli such as interleukin 6, and decreased by iron deficiency, enhanced erythropoiesis and hypoxia. It is supposed to play a crucial role in changes of iron metabolism in anaemia of chronic disease, which is characterized by sequestering iron in macrophages and decreasing its availability for red blood cell production. To study the effect of tumour growth on hepcidin expression, we implanted human melanoma cells into mice and studied the changes of the amount of liver hepcidin mRNA by real-time PCR. We observed development of anaemia, which correlated with the size of the tumour. Hepcidin expression significantly decreased with the anaemia development, but in late stages we observed an increase of its expression together with an increase of mRNA for interleukin 6. However, the increase of hepcidin expression could be inhibited by exogenous erythropoietin administration. In our model of tumour growth, hepcidin expression reflected anaemia development and iron deficiency, erythropoietin administration and inflammation, and we suppose that it could therefore serve as a useful marker of these clinical situations common in cancer patients and play a role in the pathogenesis of cancer-associated anaemia.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Eritropoetina/administração & dosagem , Inflamação/patologia , Deficiências de Ferro , Fígado/metabolismo , Melanoma/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Anemia/complicações , Anemia/patologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Proliferação de Células/efeitos dos fármacos , Eritropoetina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hepcidinas , Humanos , Inflamação/complicações , Injeções Subcutâneas , Interleucina-6/genética , Interleucina-6/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Melanoma/complicações , Melanoma/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Hepcidin is a key regulator of iron homeostasis, while hemojuvelin is an important component of the hepcidin regulation pathway. It has been recently proposed that soluble hemojuvelin, produced from hemojuvelin by the protease furin, decreases hepcidin expression. The aim of the presented study was to examine the downregulation of hepcidin by chronic bleeding in hemojuvelin-mutant mice. Male mice with targeted disruption of the hemojuvelin gene (Hjv-/- mice) and wild-type littermates were maintained on an iron-deficient diet and subjected to weekly phlebotomies for 7 weeks. Gene expression was examined by real-time PCR. In wild type mice, repeated bleeding decreased hepcidin mRNA by two orders of magnitude. In Hjv-/- mice, basal hepcidin expression was low; however, repeated bleeding also decreased hepcidin mRNA content by an order of magnitude. Phlebotomies reduced hepatic iron overload in Hjv-/- mice by 80 %. Liver and muscle furin mRNA content was not significantly changed. No effect on hepatic Tmprss6 mRNA content was observed. Results from the study indicate that soluble hemojuvelin is not the sole factor responsible for hepcidin downregulation. In addition, the presented data suggest that, under in vivo conditions, tissue hypoxia does not transcriptionally regulate the activity of furin or TMPRSS6 proteases.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Eritropoese , Hemorragia/metabolismo , Proteínas de Membrana/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Hipóxia Celular , Modelos Animais de Doenças , Regulação para Baixo , Furina/metabolismo , Proteínas Ligadas por GPI , Proteína da Hemocromatose , Hemorragia/etiologia , Hemorragia/genética , Hepcidinas , Deficiências de Ferro , Sobrecarga de Ferro/metabolismo , Sobrecarga de Ferro/prevenção & controle , Ferro da Dieta/administração & dosagem , Fígado/metabolismo , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Flebotomia , RNA Mensageiro/metabolismo , Serina Endopeptidases/metabolismo , Fatores de TempoRESUMO
Hepcidin, a key regulator of iron metabolism, plays a crucial role in the pathogenesis of anemia of chronic disease. Although it is produced mainly in the liver, its recently described expression in adipose tissue has been shown to be enhanced in massive obesity due to chronic low-grade inflammation. Our objective was to study the changes in hepcidin expression in adipose tissue during acute-phase reaction. We measured hepcidin mRNA expression from isolated subcutaneous and epicardial adipose tissue at the beginning and at the end of the surgery. The expression of mRNAs for hepcidin and other iron-related genes (transferrin receptor 1, divalent metal transporter 1, ferritin, ferroportin) were measured by real-time RT-PCR. Hepcidin expression significantly increased at the end of the surgery in subcutaneous but not in epicardial adipose tissue. Apart from the increased levels of cytokines, the parameters of iron metabolism showed typical inflammation-induced changes. We suggest that acute inflammatory changes could affect the regulation of hepcidin expression in subcutaneous adipose tissue and thus possibly contribute to inflammation-induced systemic changes of iron metabolism.
Assuntos
Reação de Fase Aguda/metabolismo , Tecido Adiposo/metabolismo , Peptídeos Catiônicos Antimicrobianos/genética , Procedimentos Cirúrgicos Cardíacos , Gordura Subcutânea/metabolismo , Reação de Fase Aguda/etiologia , Reação de Fase Aguda/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Índice de Massa Corporal , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Procedimentos Cirúrgicos Eletivos , Feminino , Hepcidinas , Humanos , Mediadores da Inflamação/sangue , Ferro/sangue , Masculino , Pessoa de Meia-Idade , Pericárdio , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Fatores de Tempo , Regulação para CimaRESUMO
Hepcidin is a key regulator of iron metabolism and plays an important role in many pathologies. It is increased by iron administration and by inflammation, while erythropoiesis downregulates its expression. It decreases iron availability and thus contributes to anemia of chronic diseases. The aim of the study was to measure hepcidin as a marker and pathogenetic factor in ANCA-associated vasculitis (AAV). Hepcidin plasma concentration was measured by the immunological method in 59 patients with AAV and compared to patients with non-vasculitic etiology of chronic kidney disease, patients on hemodialysis (HD), with systemic lupus erythematodes (SLE) and to healthy controls and blood donors, and was correlated with the parameters of iron metabolism, inflammation, activity of the process and kidney function. Hepcidin concentration was increased in patients with AAV, SLE and HD and correlated positively with C-reactive protein, serum ferritin and creatinine, and negatively with hemoglobin and serum transferrin. In active form of AAV it correlated with the clinical scoring system (BVAS). Hepcidin can thus be considered as a pathogenetic factor of anemia in AAV and can be used for evaluation of inflammation in AAV and as an additional marker in active forms of the disease.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/sangue , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/diagnóstico , Hepcidinas/sangue , Adulto , Idoso , Anemia/sangue , Anemia/diagnóstico , Biomarcadores/sangue , Feminino , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/diagnóstico , Masculino , Pessoa de Meia-Idade , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/diagnósticoRESUMO
Proteinuria is often used as a surrogate marker in monitoring and predicting outcome in patients with chronic kidney diseases, but it is non-specific. IgAN belongs to the most common primary glomerulonephritis worldwide with serious prognosis. The main aim of this work was to assess differences in urine proteins in patients with IgA nephropathy and to identify abnormal proteins as potential biomarkers of IgA nephropathy or the renal disease. In our pilot project, we selected 20 patients and compared them with 20 healthy volunteers. Protein quantification was performed using iTRAQ (isobaric tag for relative and absolute quantitation) labeling method. The peptides were separated by the isoelectric focusing method (IEF) and nano-LC with C18 column and identified by mass spectrometry using MALDI-TOF/TOF MS. Proteins´ lists obtained from IEF-LC-MS-MS/MS analysis were combined and contained 201 proteins. It was found out that 113 proteins were common in both experiments. 30 urinary proteins were significantly up- or down-regulated in patients with IgA nephropathy. We characterized potential biomarkers such as alpha-1-antitrypsin, apolipoprotein A-I, CD44 antigen or kininogen. Potential biomarkers of IgAN should be validated in further studies.
Assuntos
Glomerulonefrite por IGA/genética , Glomerulonefrite por IGA/urina , Proteômica/métodos , Adulto , Idoso , Apolipoproteína A-I/genética , Apolipoproteína A-I/urina , Biomarcadores/urina , Feminino , Glomerulonefrite por IGA/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Adulto Jovem , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/urinaRESUMO
Hepcidin, a key regulator of iron metabolism, decreases intestinal absorption of iron and its release from macrophages. Iron, anemia, hypoxia, and inflammation were reported to influence hepcidin expression. To investigate regulation of the expression of hepcidin and other iron-related genes, we manipulated erythropoietic activity in mice. Erythropoiesis was inhibited by irradiation or posttransfusion polycythemia and stimulated by phenylhydrazine administration and erythropoietin. Gene expression of hepcidin and other iron-related genes (hemojuvelin, DMT1, ferroportin, transferrin receptors, ferritin) in the liver was measured by the real-time polymerase chain reaction. Hepcidin expression increased despite severe anemia when hematopoiesis was inhibited by irradiation. Suppression of erythropoiesis by posttransfusion polycythemia or irradiation also increased hepcidin mRNA levels. Compensated hemolysis induced by repeated phenylhydrazine administration did not change hepcidin expression. The decrease caused by exogenous erythropoeitin was blocked by postirradiation bone marrow suppression. The hemolysis and anemia decrease hepcidin expression only when erythropoiesis is functional; on the other hand, if erythropoiesis is blocked, even severe anemia does not lead to a decrease of hepcidin expression, which is indeed increased. We propose that hepcidin is exclusively sensitive to iron utilization for erythropoiesis and hepatocyte iron balance, and these changes are not sensed by other genes involved in the control of iron metabolism in the liver.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Eritropoese , Hemólise , Fígado/metabolismo , RNA Mensageiro/metabolismo , Anemia Hemolítica/induzido quimicamente , Anemia Hemolítica/metabolismo , Anemia Hemolítica/fisiopatologia , Animais , Proteínas de Transporte de Cátions/metabolismo , Eritropoese/efeitos dos fármacos , Eritropoese/efeitos da radiação , Eritropoetina/farmacologia , Ferritinas/metabolismo , Proteínas Ligadas por GPI , Raios gama , Regulação da Expressão Gênica , Proteína da Hemocromatose , Hemólise/efeitos dos fármacos , Hemólise/efeitos da radiação , Hepcidinas , Compostos de Ferro/metabolismo , Fígado/efeitos dos fármacos , Fígado/efeitos da radiação , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fenil-Hidrazinas , Policitemia/metabolismo , Policitemia/fisiopatologia , Receptores da Transferrina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Haem carrier protein 1 (Hcpl) is a component of the haem-iron uptake pathway in the small intestine. Using quantitative real-time PCR, we examined the expression of Hcp1 and other intestinal iron-transporting proteins in male C57BL/6 mice with experimentally altered iron homeostasis. Intestinal Hcp1 mRNA content was not significantly changed by iron overload (600 mg/kg); however, it was increased to 170 % of controls 72 h after withdrawal of 0.7 ml of blood; the same treatment increased intestinal Cybrd1 mRNA to 900 % of controls. LPS treatment (1 mg/kg, 6 h) decreased intestinal Hcp1 mRNA content to 66 % of controls and Flvcr mRNA content to 65 % of controls, while Cybrd1 mRNA, Dmt1 mRNA and Fpn1 mRNA decreased to 6 %, 43 % and 32 %, respectively. In 129SvJ mice with targeted disruption of the hemojuvelin (Hfe2) gene, which display very low expression of liver hepcidin, Cybrd1 mRNA content increased to 1040 %, Dmt1 mRNA content to 200 % and Fpn1 mRNA to 150 % when compared to wild-type mice; changes in Hcp1, Abcg2 and Flver mRNA content were only minor. Overall, these results suggest that, during inflammation, the intestinal haem-iron uptake pathway is not as strongly transcriptionally downregulated as the non-haem iron uptake pathway. A decrease in circulating hepcidin increases the expression of proteins participating in non-haem iron uptake, but has no significant effect on Hcp1 mRNA content.
Assuntos
Heme/metabolismo , Sobrecarga de Ferro/metabolismo , Ferro/metabolismo , Lipopolissacarídeos/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas Ligadas por GPI , Proteína da Hemocromatose , Hepcidinas , Deficiências de Ferro , Fígado/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Transportador de Folato Acoplado a Próton , RNA Mensageiro/metabolismoRESUMO
The effect of the protoporphyrinogen oxidase-inhibiting herbicide fomesafen on liver porphyrin accumulation was studied in long-term high-dose experiments. Fomesafen caused liver accumulation of uroporphyrin and heptacarboxylic porphyrin when fed at 0.25% in the diet to male ICR mice for 5 months (fomesafen-treated mice: 52 nmol uroporphyrin, 21 nmol heptacarboxylic porphyrin/g liver; control mice: traces of uroporphyrin, heptacarboxylic porphyrin not detected). Uroporphyrinogen decarboxylase activity was depressed to about 25% of control values. Iron treatment accelerated the development of this porphyria cutanea tarda-like experimental porphyria both in ICR and C57B1/6J mice. In contrast to other uroporphyrinogen decarboxylase inhibitors, fomesafen treatment did not increase the cytochrome P450IA-related activities and the amount of P450IA2 protein was shown to be significantly decreased by Western immunoblotting. Thus, fomesafen is a unique chemical that inhibits both the oxidation of protoporphyrinogen as well as the conversion of uroporphyrinogen to coproporphyrinogen. However, the accumulation of highly carboxylated porphyrins is evident only after prolonged treatment with high doses of the herbicide.
Assuntos
Benzamidas/farmacologia , Herbicidas/farmacologia , Fígado/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Oxirredutases/antagonistas & inibidores , Porfirinas/química , Uroporfirinas/química , Animais , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Flavoproteínas , Éteres Difenil Halogenados , Rim/química , Rim/efeitos dos fármacos , Fígado/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Proteínas Mitocondriais , Éteres Fenílicos/farmacologia , Protoporfirinogênio OxidaseRESUMO
Iron is an essential element and its amount and balance must be precisely regulated. Iron intestinal absorption is essential for the iron balance; however, the precise mechanism of its regulation remains unknown. Antimicrobial peptide hepcidin, produced in the liver, is considered as a key regulator of iron absorption and kinetics in the organism. Its expression increases in response to the iron overload. Hepcidin decreases iron absorption in the duodenum and causes its sequestration in macrophages. Apart from the iron, inflammation increases hepcidin expression in the liver, and hepcidin is considered to be acute phase protein. Hepcidin is not only the physiological regulator of iron kinetics but is supposed to be a part of the pathogenetic mechanism of anaemia accompanying chronic diseases and its relationship to the hereditary hemochromatosis is also studied.
Assuntos
Peptídeos Catiônicos Antimicrobianos/fisiologia , Ferro/metabolismo , Proteínas de Fase Aguda/fisiologia , Anemia/fisiopatologia , Hemocromatose/genética , Hemocromatose/fisiopatologia , Hepcidinas , Humanos , Inflamação/fisiopatologia , Absorção Intestinal/fisiologiaRESUMO
Matriptase-2, a membrane protein encoded by the Tmprss6 gene, is a negative regulator of hepcidin expression. Although matriptase-2 has been proposed to cleave membrane hemojuvelin, we have recently found decreased hemojuvelin protein levels in Tmprss6 -/- mice. The purpose of this study was to confirm this observation by determining hemojuvelin protein levels in another strain of mice with disrupted Tmprss6 gene, and to determine the effect of matriptase-2 deficiency on the expression of other membrane proteins participating in the bone morphogenetic protein signal transduction. Mask mice, which lack the proteolytic domain of matriptase-2, displayed decreased liver hemojuvelin protein content, while Id1 mRNA level, an indicator of hemojuvelin-dependent signal transduction, was increased. Protein levels of bone morphogenetic protein receptors Alk3 and Acvr2a were unchanged, and transferrin receptor 2 and neogenin protein levels were slightly decreased. The results confirm that the loss of matriptase-2 increases bone morphogenetic protein-dependent signaling, while paradoxically decreasing liver hemojuvelin protein content. The regulation of transferrin receptor 2 protein levels by transferrin saturation was not affected in mask mice. How the loss of matriptase-2 proteolytic activity leads to decreased hemojuvelin protein levels is at present unclear.
Assuntos
Fígado/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Serina Endopeptidases/deficiência , Receptores de Activinas Tipo II/metabolismo , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Regulação para Baixo , Feminino , Proteínas Ligadas por GPI , Proteína da Hemocromatose , Proteína 1 Inibidora de Diferenciação/genética , Proteína 1 Inibidora de Diferenciação/metabolismo , Injeções Intraperitoneais , Deficiências de Ferro , Complexo Ferro-Dextran/administração & dosagem , Fígado/efeitos dos fármacos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/metabolismo , Receptores da Transferrina/metabolismo , Serina Endopeptidases/genéticaRESUMO
Hepcidin, a recently discovered antimicrobial peptide synthesized in the liver, was identified to be the key mediator of iron metabolism and distribution. Despite our knowledge of hepcidin increased in recent years, there are only limited data on hepcidin regulation during systemic inflammatory response in human subjects. In a prospective study, the time course of plasma hepcidin was analyzed in relations to six inflammatory parameters - plasma cytokines and acute-phase proteins in patients undergoing uncomplicated pulmonary endarterectomy. Twenty-four patients (males, aged 52.6+/-10.2 years, treated with pulmonary endarterectomy in a deep hypothermic circulatory arrest) were enrolled into study. Hepcidin, interleukin (IL)-6, IL-8, tumor necrosis factor-alpha, C-reactive protein, alpha(1)-antitrypsin and ceruloplasmin arterial concentrations were measured before surgery and repeatedly within 120 h post-operatively. Hemodynamic parameters, hematocrit and markers of iron metabolism were followed up. In a postoperative period, hepcidin increased from preoperative level 8.9 ng/ml (6.2-10.7) (median and interquartile range) to maximum 16.4 ng/ml (14.1-18.7) measured 72 h after the end of surgery. Maximum post-operative concentrations of hepcidin correlated positively with maximum IL-6 levels. Both hepcidin and IL-6 maximum concentrations correlated positively with extracorporeal circulation time. In conclusions, the study demonstrated that plasma hepcidin is a positive acute-phase reactant in relation to an uncomplicated large cardiac surgery. Hepcidin increase was related to IL-6 concentrations and to the duration of surgical procedure. Our clinical findings are in conformity with recent experimental studies defining hepcidin as a type II acute-phase protein.
Assuntos
Peptídeos Catiônicos Antimicrobianos/sangue , Interleucina-6/sangue , Artéria Pulmonar/cirurgia , Proteínas de Fase Aguda/metabolismo , Endarterectomia , Hepcidinas , Humanos , Hipertensão Pulmonar/terapia , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
The effect of peroxidizing herbicides on the liver porphyrin content of experimental animals was examined. Mice treated with the herbicide oxadiazon accumulated uroporphyrin and protoporphyrin in the liver. Formesafen-treated mice accumulated uroporphyrin and heptacarboxylic porphyrin.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Herbicidas/efeitos adversos , Fígado/química , Porfirias/induzido quimicamente , Porfirinas/análise , Animais , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos ICR , Porfirias/metabolismoRESUMO
The effects of the herbicides fomesafen, oxyfluorfen, oxadiazon and fluazifop-butyl on porphyrin accumulation in mouse liver, rat primary hepatocyte culture and HepG2 cells were investigated. Ten days of herbicide feeding (0.25% in the diet) increased the liver porphyrins in male C57B1/6J mice from 1.4 +/- 0.6 to 4.8 +/- 2.1 (fomesafen) 16.9 +2- 2.9 (oxyfluorfen) and 25.9 +/- 3.1 (oxadiazon) nmol/g wet weight, respectively. Fluazifop-butyl had no effect on liver porphyrin metabolism. Fomesafen, oxyfluorfen and oxadiazon increased the cellular porphyrin content of rat hepatocytes after 24 h of incubation (control, 3.2 pmol/mg protein, fomesafen, oxyfluorfen and oxadiazon at 0.125 mM concentration 51.5, 54.3 and 44.0 pmol/mg protein, respectively). The porphyrin content of HepG2 cells increased from 1.6 to 18.2, 10.6 and 9.2 pmol/mg protein after 24 h incubation with the three herbicides. Fluazifop-butyl increased hepatic cytochrome P450 levels and ethoxy- and pentoxyresorufin O-dealkylase (EROD and PROD) activity, oxyfluorfen increased PROD activity. Peroxisomal palmitoyl CoA oxidation increased after fomesafen and fluazifop treatment to about 500% of control values both in mouse liver and rat hepatocytes. Both rat hepatocytes and HepG2 cells can be used as a test system for the porphyrogenic potential of photobleaching herbicides.
Assuntos
Herbicidas/toxicidade , Fígado/efeitos dos fármacos , Oxidiazóis/toxicidade , Porfirinas/biossíntese , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Di-Hidropiridinas/toxicidade , Éteres Difenil Halogenados , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Éteres Fenílicos/toxicidade , Ratos , Ratos Wistar , Células Tumorais CultivadasRESUMO
The Harderian gland of rodents is the only known tissue with physiological occurrence of high concentrations of porphyrins. In this report we describe the occurrence of considerable concentrations of porphyrins in the extra-orbital and intra-orbital lacrimal glands of the male rat. Similarly as in the Harderian gland, HPLC analysis revealed protoporphyrin as being the prevalent porphyrin homologue in the lacrimal glands. The results may contribute to elucidation of the yet unknown function of the Harderian gland and its porphyrins.
Assuntos
Glândula de Harder/análise , Aparelho Lacrimal/análise , Porfirinas/análise , Envelhecimento/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Cricetinae , Masculino , Ratos , Ratos EndogâmicosRESUMO
Administration of oxadiazon or oxyfluorfen (1000 ppm in the diet) to male BALB/c mice for 9 days resulted in experimental porphyria, resembling the acute phase of human variegate porphyria. Urinary concentrations of 5-aminolevulinic acid and porphobilinogen reached 1500 and 3000 mumol/L, respectively. Both herbicides caused a decrease of protoporphyrinogen oxidase activity in liver and kidney. Brain protoporphyrinogen oxidase activity was not altered. Liver and kidney porphyrin content increased to 11 and 17 nmol/g, respectively (control mice, 2 nmol/g). Over 50% of liver and kidney porphyrins were in the reduced (porphyrinogen) form. Bile of oxadiazon-treated mice contained 700 nmol/mL of protoporphyrinogen (control mice, 15 nmol/mL). Porphyrin content of the trigeminal nerve increased from 1 nmol/g in control animals to 11 nmol/g in oxadiazon-treated animals, suggesting a possible contribution of peripheral nerve porphyrins to porphyric neuropathy. Mice treated with 125 ppm of oxadiazon in the diet for 9 days excreted moderately elevated levels of porphobilinogen in urine (control mice, less than 50 mumol/L; treated mice, 330 mumol/L). Administration of phenobarbital or phenytoin (single injections on days 7, 8, and 9) increased the urinary porphobilinogen concentration to 3500 mumol/L. This response to porphyrogenic drugs resembles the response observed in human acute porphyrias.
Assuntos
Herbicidas/toxicidade , Porfirias/induzido quimicamente , Porfirinogênios/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/biossíntese , Indução Enzimática/efeitos dos fármacos , Éteres Difenil Halogenados , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Oxidiazóis/toxicidade , Éteres Fenílicos/farmacologia , Porfobilinogênio/urina , Porfirias/metabolismo , Porfirias/patologia , Porfirinas/urinaRESUMO
The rapid identification of mycobacterial DNA in clinical samples by PCR can be useful in the diagnosis of tuberculous infections, but several large studies have found that the sensitivity of this approach is not better than that of culture. In order to improve the sensitivity of detection of mycobacterial DNA in clinical specimens from patients with paucibacillary forms of tuberculosis, we have developed a procedure permitting the specific capture of mycobacterial DNA in crude samples prior to amplification, thereby concentrating the target sequences and removing irrelevant DNA and other potential inhibitors of the amplification reaction (sequence capture-PCR). By using this approach to capture and amplify two different sequences specific for organisms of the Mycobacterium tuberculosis complex (IS6110 and the direct repeat region), it was possible to detect as little as one genome of mycobacterial DNA in samples containing up to 750 micrograms of total DNA, representing a 10- to 100-fold increase in sensitivity compared with that obtained by purifying total DNA prior to amplification. Detection of the IS6110 sequence in pleural fluid samples from patients with tuberculous pleurisy by sequence capture-PCR gave positive results in 13 of 17 cases, including 3 of 3 culture-positive samples and 10 of 14 culture-negative samples. In contrast, when total DNA was purified from these samples by adsorption to a silica matrix prior to amplification, only the three culture-positive samples were positive by PCR. The sensitivity of detection of the direct repeat sequence in these samples by sequence capture-PCR was similar to that of IS6110 and, in addition, permitted immediate typing of the strains from some patients. We conclude that sequence capture-PCR improves the sensitivity of detection of mycobacterial DNA in paucibacillary samples. This approach should be useful in detecting rare target sequences from organisms implicated in other pathologic processes.
Assuntos
DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Adulto , Técnicas de Tipagem Bacteriana , Sequência de Bases , Elementos de DNA Transponíveis , Estudos de Avaliação como Assunto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mycobacterium tuberculosis/classificação , Derrame Pleural/microbiologia , Reação em Cadeia da Polimerase/estatística & dados numéricos , Sequências Repetitivas de Ácido Nucleico , Sensibilidade e Especificidade , Tuberculose/diagnóstico , Tuberculose/microbiologia , Tuberculose Pleural/diagnóstico , Tuberculose Pleural/microbiologiaRESUMO
To evaluate the role of mycobacterial infection in the pathogenesis of sarcoidosis, several groups have attempted to identify mycobacterial DNA in clinical samples from these patients by polymerase chain reaction (PCR), but widely divergent results have been obtained. It has been suggested that differences in the sensitivity of the procedures used may explain these discrepant results. To test this possibility, the presence of mycobacterial DNA was sought in biopsies from patients with sarcoidosis using sequence capture-PCR, a procedure that is 100-fold more sensitive in detecting mycobacterial DNA in paucibacillary samples than standard PCR protocols. Using this approach, DNA corresponding to two different sequences specific for organisms of the Mycobacterium tuberculosis complex (the 1S6110 insertion element and the DR region) could not be detected in any of the 15 biopsies from patients with sarcoidosis, whereas a high proportion of positive results was obtained for tissue biopsies and other clinical samples from patients with active tuberculosis, including samples that were smear-negative/culture-positive and smear-negative/culture-negative. These results support prior studies suggesting that M. tuberculosis does not play a pathogenic role in sarcoidosis in most patients.