RESUMO
It is expensive to induce experimental autoimmune myasthenia gravis (EAMG) by active immunity, and difficult to obtain natural acetylcholine receptor (AChR). We sought a new method of inducing EAMG by immunizing rats with artificially synthesized AChR. The AChR mRNA in TE671 cells was extracted and reverse transcribed. The inclusion body was purified and protein concentration was determined, and the EAMG animal model was used for induction. The serum was extracted from rat blood. The antibody titer was determined using enzyme-linked immunosorbant assay (ELISA). The concentration of decay accelerating factor (DAF) in the rat serum was determined by ELISA, and the metabolism of serum rDAF was determined by western blot. We evaluated the inhibition of rDAF by determining the 50% complement hemolysis unit in the rat serum. The extracellular domain (ECD) nucleotide sequence clone produced by polymerase chain reaction was completely consistent with that in the human gene bank; it was induced by isopropyl ß-D-1-thiogalactopyranoside to express the protein after insertion into vector pET16b. Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the inclusion body protein was the exact target. The ECD protein was able to bind with mAb35 after dialysis and renaturation, which demonstrated protein activity. The soluble ECD protein was used to immunize rats and obtain the EAMG models. The inhibitory effect of the complement was unsatisfactory owing to high decay rate after rDAF injection into the EAMG models. It is easy to induce the EAMG model by obtaining the AChRTEα1 subunit ECD protein using the substitution method.
Assuntos
Antígenos CD55/uso terapêutico , Miastenia Gravis Autoimune Experimental/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Animais , Antígenos CD55/administração & dosagem , Antígenos CD55/sangue , Modelos Animais de Doenças , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Injeções Intravenosas , Miastenia Gravis Autoimune Experimental/sangue , Miastenia Gravis Autoimune Experimental/patologia , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/farmacologia , Renaturação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Ratos Endogâmicos Lew , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
CD55 and CD59 are glycosylphosphatidylinositol-anchored proteins with complement inhibitory properties. CD55 inhibits the formation of C3 convertases, and CD59 prevents the terminal polymerisation of the membrane attack complex. It has been reported that SLE patients seems to have an acquired deficiency of these proteins associated with secondary autoimmune haemolytic anaemia and lymphopenia. The aim of this study was to evaluate the presence of altered CD55 and CD59 expression on peripheral blood cells from SLE patients. Flow cytometric analyses were performed on red and white blood cells from 23 SLE patients and 23 healthy controls. We observed more CD55- and CD59-lymphocytes (p=0.005 and p=0.019, respectively), and CD59-granulocytes (p=0.045) in SLE patients than in controls. These results suggest there is an altered pattern of CD55 and CD59 expression on the peripheral blood cells of SLE patients, and it may play a role in the cytopenias in these patients.
Assuntos
Antígenos CD55/sangue , Antígenos CD59/sangue , Lúpus Eritematoso Sistêmico/sangue , Adulto , Anemia Hemolítica Autoimune/sangue , Anemia Hemolítica Autoimune/etiologia , Contagem de Células Sanguíneas , Células Sanguíneas/imunologia , Células Sanguíneas/patologia , Feminino , Citometria de Fluxo , Glicosilfosfatidilinositóis/sangue , Glicosilfosfatidilinositóis/imunologia , Humanos , Imunofenotipagem , Lúpus Eritematoso Sistêmico/complicações , Lúpus Eritematoso Sistêmico/imunologia , Linfopenia/sangue , Linfopenia/etiologia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
The regulatory proteins CD55 and CD59 are glycolsylphosphatidylinositol-anchored, type I cell surface proteins, which inhibit formation of the C3 convertases and prevent the terminal polymerization of the membrane attack complexes, respectively. Paroxysmal nocturnal hemoglobinuria (PNH) is a genetic disorder due to the impaired conformation of the glycolsylphosphatidylinositol anchor, which results in the deficient expression of CD55 and CD59 leading to excessive destruction of red cells and leukocytes. We have studied the expression of these two molecules in red blood cells, granulocytes and platelets in patients with PNH (two patients), autoimmune hemolytic anemia (AIHA) (seven patients), autoimmune thrombocytopenia (ATP) (22 patients), systemic lupus erythematosus (SLE) (19 patients), aplastic anemia (AA) (eight patients), and Evans syndrome (ES) (two patients). A diminished expression of CD55 and CD59 was found in the three cell lines of the two patients with PNH. In the seven patients with AIHA two were found with CD59 diminished expression in red blood cells and one with CD59 diminished expression in granulocytes. In the patients with ATP one was found with CD55 diminished expression in red blood cells, one with CD59 diminished expression granulocytes and one with a CD59 diminished expression in the platelets. In the subset of patients with SLE only one was found with a CD55 diminished expression in the red blood cells. In the patients with AA, a diminished expression in red blood cells was not found; however, one patient was found with a diminished expression of CD59 in granulocytes, and one patient with a diminished expression of CD55 in the platelets. In the two patients with ES we did not found changes in the expression of CD55 and CD59. We conclude that the diminished expression of the glycolsylphosphatidylinositol-anchored type I cell surface proteins CD55 and CD59 is not specific to PNH and that it can be found in patients with a variety of autoimmune disorders. Additional studies are needed to define the role of the deficiencies of CD55 and CD59 in the pathogenesis of autoimmune hemocytopenias.
Assuntos
Antígenos CD55/biossíntese , Antígenos CD59/biossíntese , Hemoglobinúria Paroxística/imunologia , Anemia Hemolítica Autoimune/sangue , Anemia Hemolítica Autoimune/imunologia , Plaquetas/imunologia , Antígenos CD55/sangue , Antígenos CD59/sangue , Linhagem Celular , Citometria de Fluxo , Granulócitos/imunologia , Hemoglobinúria Paroxística/sangue , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Púrpura Trombocitopênica Idiopática/sangue , Púrpura Trombocitopênica Idiopática/imunologiaRESUMO
CD55 and CD59 are glycosylphosphatidylinositol-anchored proteins with complement inhibitory properties. Autoimmune hemolytic anemia (AIHA) has been associated with antiphospholipid antibodies (APLA). The aim of this study was to evaluate the presence of APLA and its possible correlation with diminished CD55 and CD59 in red blood cells from patients with primary AIHA or secondary to systemic lupus erythematosus (SLE). Flow cytometric analyses were performed on CD55 and CD59 stained erythrocytes from 24 patients (primary AIHA, n=8; AIHA plus SLE, n=11; and SLE without AIHA, n=5) and 20 healthy controls. Antibodies to several phospholipids were detected in the sera by ELISA. Most patients with AIHA plus SLE and few with primary AIHA showed deficiency of either or both CD55 and CD59 expression and was not associated to the presence of APLA, while SLE patients exhibited a normal expression of these molecules. Although our findings showed CD55 and CD59 deficiency in primary or secondary AIHA, it appears that this defect plays a facilitator rather than a triggering role for the hemolytic process. Additionally, a role of anti-phospholipid antibodies as causative of this acquired defect is questionable.
Assuntos
Antígenos CD55/sangue , Antígenos CD55/imunologia , Antígenos CD59/sangue , Antígenos CD59/imunologia , Eritrócitos/metabolismo , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Adulto , Anemia Hemolítica Autoimune/sangue , Anemia Hemolítica Autoimune/imunologia , Estudos de Casos e Controles , Eritrócitos/imunologia , Feminino , Citometria de Fluxo , Hemólise , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
The expansion of paroxysmal nocturnal hemoglobinuria (PHN) clone was evaluated in a patient with aplastic anemia (AA) of 18 years of evolution during an hemolytic crisis. On day 0, Ham and Sucrosa tests were positive and hematological parameters were altered. Low hemoglobin (Hb) levels and erythrocyte and leukocyte counts were found and continued decreasing on days 7 and 24 (last day of study). High LDH levels, indirect bilirubin and reticulocyte counts were detected throughout. We evaluated CD55 and CD59 on erythrocytes by flow cytometry. Our results showed low CD55 expression with respect to the normal pattern. Since day 0, CD59 staining detected two red cell populations: PNH I (48%), cells with positive fluorescence similar to normal and PNH III (52%), negative cells (PNH clone). These negative cells increased, reaching 70% on day 24. Other membrane anchored leukocyte proteins were also absent (CD14) or decreased (CD16). We found a good correlation between clinical observations, evolution of the laboratory values and expansion of the PNH clone.
Assuntos
Anemia Aplástica/sangue , Antígenos CD59/sangue , Eritrócitos/imunologia , Citometria de Fluxo/métodos , Hemoglobinúria Paroxística/sangue , Adulto , Anemia Aplástica/diagnóstico , Anemia Aplástica/imunologia , Antígenos CD55/sangue , Células Clonais/imunologia , Feminino , Hemoglobinúria Paroxística/diagnóstico , Hemoglobinúria Paroxística/imunologia , Humanos , Leucócitos/imunologia , Proteínas de Membrana/sangueRESUMO
Se estudió por citometría de flujo la expansión de un clon HPN durante una crisis hemolítica en una paciente con anemia aplásica (AA) de 18 años de evolución. Cuando las pruebas de Ham y Sacarosa fueron positivas (día 0) estaban disminuidos los niveles de hemoglobina y los recuentos de eritrocitos y leucocitos. Estos descensos se fueron acentuando hasta el último día del estudio (día 24). Durante este período los niveles de LDH, bilirrubina indirecta y reticulocitos se mantuvieron elevados. El clon HPN se investigó evaluando la expresión de CD55 y CD59 sobre los eritrocitos. Las células de la paciente presentaron una menor intensidad de fluorescencia para CD55 respecto del control normal. Con la marcación con anti CD59 se detectó la presencia de dos poblaciones de hematíes: HPN I, con fluorescencia positiva semejante a la de la población normal y HPN III, con fluorescencia negativa, correspondiente al clon HPN. El porcentaje de la población HPN III se incrementó de 52 por ciento (días 0 y 7) a 70 por ciento (día 24). Las proteínas de la membrana leucocitaria también se encontraron afectadas (CD14 ausente y CD16 disminuida). El análisis global de los datos reveló una buena correlación entre la observación clínica, la evolución de los valores del laboratorio y la expansión del clon. (AU)
Assuntos
Humanos , Feminino , Adulto , Hemoglobinúria Paroxística/sangue , Anemia Aplástica/sangue , Citometria de Fluxo/métodos , Glicoproteínas de Membrana/sangue , Eritrócitos/imunologia , Hemoglobinúria Paroxística/diagnóstico , Hemoglobinúria Paroxística/imunologia , Anemia Aplástica/diagnóstico , Anemia Aplástica/imunologia , Anemia Hemolítica/sangue , Anemia Hemolítica/diagnóstico , Anemia Hemolítica/imunologia , Antígenos CD55/sangue , Antígenos CD59/sangue , Proteínas de Membrana/sangue , Leucócitos/imunologia , Células Clonais/imunologiaRESUMO
Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by total or partial deficiency of membrane proteins anchored to the cell surface through a glycosylphosphatidyl-inositol (GPI) moiety. The relationship between the size of the PNH clone, determined by the expression of GPI-anchored proteins (AP; CD14, CD48, CD55, CD59, and CD66b) on erythrocytes, lymphocytes, monocytes, and granulocytes using forward and side scatter analysis, and severity of the disease was evaluated in 19 PNH patients. CD55 antigen expression did not delineate abnormal erythrocytes as well as did anti-CD59. The proportion of monocytes deficient in CD55, CD59, CD48, and CD14 (48-97%) and of granulocytes deficient in CD55, CD59, and CD66b (60-99%) was greater than the proportion of erythrocytes deficient in CD59 (24-95%) and the proportion of lymphocytes deficient in CD55 and CD59 (30-98%). There were no significant correlations among reticulocyte, leukocyte, and platelet counts and GPI-AP-deficient immunophenotypes in red and white blood cells. However, high coefficients of determination were seen between hemoglobin levels and granulocytes deficient in CD59 (r(2) = 0.76), CD55 (r(2) = 0.74), and CD66b (r(2) = 0.74) antigens and between hemoglobin and monocytes deficient in CD55 (r(2) = 0.73), CD59 (r(2) = 0.80), and CD14 (r(2) = 0.75) antigens. These results are interpreted as indicating that the size of PNH clone is better assessed by immunophenotypic analysis of monocytes and granulocytes rather than of lymphocytes and erythrocytes.
Assuntos
Antígenos de Neoplasias , Células Sanguíneas/imunologia , Células Sanguíneas/metabolismo , Proteínas Sanguíneas/metabolismo , Moléculas de Adesão Celular , Glicosilfosfatidilinositóis/sangue , Hemoglobinúria Paroxística/sangue , Hemoglobinúria Paroxística/imunologia , Adulto , Idoso , Antígenos CD/sangue , Antígeno CD48 , Antígenos CD55/sangue , Antígenos CD59/sangue , Eritrócitos/imunologia , Eritrócitos/metabolismo , Feminino , Citometria de Fluxo , Proteínas Ligadas por GPI , Granulócitos/imunologia , Granulócitos/metabolismo , Humanos , Imunofenotipagem , Receptores de Lipopolissacarídeos/sangue , Linfócitos/imunologia , Linfócitos/metabolismo , Masculino , Glicoproteínas de Membrana/sangue , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/metabolismoRESUMO
The peripheral blood cells of ten patients with biopsy-proven aplastic anemia were studied by means of flow-cytometry in order to assess the expression of two phosphatidylinositol-anchored surface proteins: CD55/DAF (decay accelerating factor) and CD59/MIRL (membrane inhibitor of reactive lysis). An abnormal expression was found in five of these ten patients, whereas the "traditional" tests for paroxysmal nocturnal hemoglobinuria (PNH) were positive only on two of these five individuals. Five of the aplastic patients were treated with anti-thymocyte globulin and cyclosporin-A and three entered a complete remission; of the latter, one had CD55/CD59 deficiencies whereas two did not. Along the study period one patient with a hemolytic pattern of PNH was identified. It is concluded that CD55 and/or CD59 abnormalities are frequent in Mexican mestizo patients with aplastic anemia, that the aplastic presentation of PNH is more frequent in Mexico than the hemolytic presentation, that the flow-cytometric identification of CPI-anchored proteins is more sensitive than the "traditional" PNH tests, and that some patients with PNH-aplasia may respond to intensive immunosuppressive treatment. The flow-cytometric identification of GPI-anchored cell surface proteins should replace the "traditional" tests in the identification of patients with PNH.
Assuntos
Anemia Aplástica/sangue , Antígenos CD55/sangue , Antígenos CD59/sangue , Indígenas Norte-Americanos , Anemia Aplástica/complicações , Anemia Aplástica/etnologia , Citometria de Fluxo , Hemoglobinúria Paroxística/sangue , Hemoglobinúria Paroxística/complicações , Hemoglobinúria Paroxística/diagnóstico , Humanos , MéxicoRESUMO
Cutaneous inoculation of Loxosceles spp. spider venoms produces local necrosis, occasionally accompanied by systemic intravascular clotting and hemolysis. In this work, we analyzed the role of the C system on the lysis of human erythrocytes (Eh) induced by Loxosceles venoms in vitro. Eh were treated with whole venom of Loxosceles laeta, Loxosceles gaucho, or Loxosceles intermedia, or with purified venom proteins, and incubated with C-sufficient (Cs-NHS) or C9-depleted autologous (C9d-NHS) serum. Hemolysis was determined spectrophotometrically, and deposition of C components or removal of C regulatory proteins was analyzed by FACS. Eh suspensions exposed to venoms or to a purified 35-kDa protein from L. intermedia were lysed after incubation with Cs-NHS, but not with C9d-NHS. Lysis was blocked by heating the serum at 50 degrees C or Ca2+/Mg2+ chelation by EDTA, but not by Ca2+ chelation with EGTA. Deposition of C1, C2, C3, C4, C5, and factor B on the venom-treated Eh occurred during activation of autologous C. Regulatory proteins decay-accelerating factor (DAF) and CD59 were not altered significantly. Conversion of C-resistant Eh into C-susceptible Eh by the L. intermedia venom was accompanied by incorporation of a 35-kDa venom protein onto the cell surface. Thirty-five-kilodalton-related proteins were detected in the two other Loxosceles venoms by ELISA, using rabbit antiserum against the L. intermedia 35-kDa protein. These data suggest that the C system mediates the lysis of human erythrocytes and, by extension, of other cell types able to incorporate the lytic factor of Loxosceles venoms on their cell surfaces.