RESUMO
BACKGROUND: There is growing evidence that neuroinflammation may contribute to schizophrenia neuropathology. Elevated pro-inflammatory cytokines are evident in the midbrain from schizophrenia subjects, findings that are driven by a subgroup of patients, characterised as a "high inflammation" biotype. Cytokines trigger the release of antibodies, of which immunoglobulin G (IgG) is the most common. The level and function of IgG is regulated by its transporter (FcGRT) and by pro-inflammatory IgG receptors (including FcGR3A) in balance with the anti-inflammatory IgG receptor FcGR2B. Testing whether abnormalities in IgG activity contribute to the neuroinflammatory abnormalities schizophrenia patients, particularly those with elevated cytokines, may help identify novel treatment targets. METHODS: Post-mortem midbrain tissue from healthy controls and schizophrenia cases (n = 58 total) was used to determine the localisation and abundance of IgG and IgG transporters and receptors in the midbrain of healthy controls and schizophrenia patients. Protein levels of IgG and FcGRT were quantified using western blot, and gene transcript levels of FcGRT, FcGR3A and FcGR2B were assessed using qPCR. The distribution of IgG in the midbrain was assessed using immunohistochemistry and immunofluorescence. Results were compared between diagnostic (schizophrenia vs control) and inflammatory (high vs low inflammation) groups. RESULTS: We found that IgG and FcGRT protein abundance (relative to ß-actin) was unchanged in people with schizophrenia compared with controls irrespective of inflammatory subtype. In contrast, FcGRT and FcGR3A mRNA levels were elevated in the midbrain from "high inflammation" schizophrenia cases (FcGRT; p = 0.02, FcGR3A; p < 0.0001) in comparison to low-inflammation patients and healthy controls, while FcGR2B mRNA levels were unchanged. IgG immunoreactivity was evident in the midbrain, and approximately 24% of all individuals (control subjects and schizophrenia cases) showed diffusion of IgG from blood vessels into the brain. However, the intensity and distribution of IgG was comparable across schizophrenia cases and control subjects. CONCLUSION: These findings suggest that an increase in the pro-inflammatory Fcγ receptor FcGR3A, rather than an overall increase in IgG levels, contribute to midbrain neuroinflammation in schizophrenia patients. However, more precise information about IgG-Fcγ receptor interactions is needed to determine their potential role in schizophrenia neuropathology.
Assuntos
Esquizofrenia , Citocinas/metabolismo , Humanos , Imunoglobulina G , Inflamação , Mesencéfalo/metabolismo , RNA Mensageiro , Receptores de IgG/genética , Receptores de IgG/metabolismo , Esquizofrenia/metabolismoRESUMO
We have identified the Xga antigen, encoded by the XG blood group gene, by employing rabbit polyclonal and mouse monoclonal antibodies raised against a peptide derived from the N-terminal domain of a candidate gene, referred to earlier as PBDX. In indirect haemagglutination assays, these anti-peptide antibodies react with Xg(a+) but not Xg(a-) erythrocytes. In antibody-specific immobilization of antigen (ASIA) and immunoblot assays, the anti-peptide antibodies react with the same molecule as does human anti-Xga. Therefore, by its identity with PBDX, Xga is identified as a cell-surface protein that is 48% homologous to CD99 (previously designated the 12E7 antigen), the product of MIC2 which is tightly linked to XG. PBDX is renamed here XG.
Assuntos
Antígenos CD , Antígenos de Grupos Sanguíneos/genética , Moléculas de Adesão Celular/genética , Genes , Antígeno 12E7 , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Western Blotting , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/fisiologia , Reações Cruzadas , Epitopos/imunologia , Fibroblastos/metabolismo , Ligação Genética , Testes de Hemaglutinação , Células-Tronco Hematopoéticas/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Coelhos , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
Low-cost, light-scattering-based particulate matter (PM) sensors are becoming more widely available and are being increasingly deployed in ambient and indoor environments because of their low cost and ability to provide high spatial and temporal resolution PM information. Researchers have begun to evaluate some of these sensors under laboratory and environmental conditions. In this study, a low-cost, particulate matter sensor (Plantower PMS 1003/3003) used by a community air-quality network is evaluated in a controlled wind-tunnel environment and in the ambient environment during several winter-time, cold-pool events that are associated with high ambient levels of PM. In the wind-tunnel, the PMS sensor performance is compared to two research-grade, light-scattering instruments, and in the ambient tests, the sensor performance is compared to two federal equivalent (one tapered element oscillating microbalance and one beta attenuation monitor) and gravimetric federal reference methods (FEMs/FRMs) as well as one research-grade instrument (GRIMM). The PMS sensor response correlates well with research-grade instruments in the wind-tunnel tests, and its response is linear over the concentration range tested (200-850 µg/m3). In the ambient tests, this PM sensor correlates better with gravimetric methods than previous studies with correlation coefficients of 0.88. However additional measurements under a variety of ambient conditions are needed. Although the PMS sensor correlated as well as the research-grade instrument to the FRM/FEMs in ambient conditions, its response varies with particle properties to a much greater degree than the research-grade instrument. In addition, the PMS sensors overestimate ambient PM concentrations and begin to exhibit a non-linear response when PM2.5 concentrations exceed 40 µg/m3. These results have important implications for communicating results from low-cost sensor networks, and they highlight the importance of using an appropriate correction factor for the target environmental conditions if the user wants to compare the results to FEM/FRMs.
Assuntos
Poluentes Atmosféricos/análise , Monitoramento Ambiental/instrumentação , Material Particulado/análise , Monitoramento Ambiental/métodos , Laboratórios , Tamanho da Partícula , Estações do Ano , VentoRESUMO
Employment of postoperative brain irradiation in the initial management of high-grade malignant glial tumors has now become standard. The addition of conventional chemotherapy to irradiation has not significantly improved median survival beyond 1 year. We treated 25 consecutive patients (13 pilot patients and 12 protocol patients) with histologically confirmed unresectable grade 3 or 4 malignant gliomas with high-dose BCNU (carmustine) followed by autologous bone marrow transplantation and whole brain irradiation. Within 3 weeks of initial surgery, each patient had autologous bone marrow stored (median 2 X 10(8) nucleated cells/kg), and then received BCNU 1,050 mg/m2 intravenously (IV). Peripheral granulocytes recovered (greater than 500/microL) at a median of 19 days (range, 10 to 37 days), and platelets recovered (greater than 20,000/microL) at a median of 18 days (range, 13 to 40 days), following bone marrow infusion. Patients received 60 Gy whole brain irradiation when granulocytes were greater than 1,500/microL. Toxicity was well tolerated. Nausea occurred in 19 patients (76%); however, only eight patients (32%) experienced vomiting (mild in three, moderate in five). Eleven patients (44%) did not require empiric antibiotics, six of whom never developed an absolute granulocyte count less than 500/microL. Three patients with a poor performance status died early (one seizure with vomiting and asphyxiation; one, klebsiella urinary tract infection (UTI) with bacteremia; one, candidal pneumonia), and one additional patient who was performing well died of pulmonary hemorrhage. The 13 pilot patients have now been followed for a median of 23 months, with a significant survival advantage compared with the 52 consecutive historical control patients who received similar surgery and radiotherapy without high-dose BCNU (P = .037). The overall study group of 25 patients also has a significant survival advantage when compared with the same historical control group, with a projected median survival of 26 months (P = .007). This new approach using early postoperative intensive therapy consisting of high-dose BCNU, autologous bone marrow transplantation, and whole brain irradiation appears to significantly improve survival.
Assuntos
Transplante de Medula Óssea , Neoplasias Encefálicas/terapia , Carmustina/administração & dosagem , Glioma/terapia , Adulto , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/cirurgia , Carmustina/efeitos adversos , Terapia Combinada , Feminino , Glioma/radioterapia , Glioma/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Náusea/induzido quimicamente , Transplante AutólogoRESUMO
Diffuse infiltrative invasion is a major cause for the dismal prognosis of glioblastoma multiforme (GBM), but the underlying mechanisms remain incompletely understood. Using human glioma stem cells (GSCs) that recapitulate the invasive propensity of primary GBM, we find that EphA2 critically regulates GBM invasion in vivo. EphA2 was expressed in all seven GSC lines examined, and overexpression of EphA2 enhanced intracranial invasion. The effects required Akt-mediated phosphorylation of EphA2 on serine 897. In vitro the Akt-EphA2 signaling axis is maintained in the absence of ephrin-A ligands and is disrupted upon ligand stimulation. To test whether ephrin-As in tumor microenvironment can regulate GSC invasion, the newly established Efna1;Efna3;Efna4 triple knockout mice (TKO) were used in an ex vivo brain slice invasion assay. We observed significantly increased GSC invasion through the brain slices of TKO mice relative to wild-type (WT) littermates. Mechanistically EphA2 knockdown suppressed stem cell properties of GSCs, causing diminished self-renewal, reduced stem marker expression and decreased tumorigenicity. In a subset of GSCs, the reduced stem cell properties were associated with lower Sox2 expression. Overexpression of EphA2 promoted stem cell properties in a kinase-independent manner and increased Sox2 expression. Disruption of Akt-EphA2 cross-talk attenuated stem cell marker expression and neurosphere formation while having minimal effects on tumorigenesis. Taken together, the results show that EphA2 endows invasiveness of GSCs in vivo in cooperation with Akt and regulates glioma stem cell properties.
Assuntos
Neoplasias Encefálicas/genética , Carcinogênese/genética , Glioblastoma/genética , Proteína Oncogênica v-akt/genética , Receptor EphA2/genética , Animais , Neoplasias Encefálicas/patologia , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Humanos , Camundongos , Camundongos Knockout , Invasividade Neoplásica/genética , Células-Tronco Neoplásicas/patologia , Proteína Oncogênica v-akt/metabolismo , Fosforilação/genética , Fatores de Transcrição SOXB1/genética , Transdução de Sinais/genéticaRESUMO
A new assay, the monoclonal antibody-immobilization of erythrocyte antigens (MAIEA) is described for the assignment of red cell antigens recognised by human allo-antisera, to particular membrane components of the red cell membrane. This technique detects tri-molecular complexes formed by the reaction of a human antibody and a mouse antibody with a particular red cell protein. A positive reaction, in an ELISA-type detection procedure, occurs if the epitopes recognised by the human and mouse antibodies are present on the same membrane component but different regions. The MAIEA assay has been developed to investigate blood group antigens and in this report its application to the study of the Lutheran, Yt and Kell blood group systems is described. The technique has been used to show that the Lu(a) and Lu3 antigens are carried on the Lutheran glycoprotein, Yta and Ytb on the enzyme acetylcholinesterase and K, k, Kpa and Kpb on the Kell glycoprotein.
Assuntos
Antígenos de Grupos Sanguíneos/imunologia , Membrana Eritrocítica/imunologia , Imunoensaio/métodos , Animais , Anticorpos Monoclonais/imunologia , Humanos , Sistema do Grupo Sanguíneo de Kell/imunologia , Sistema do Grupo Sanguíneo Lutheran/imunologia , CamundongosRESUMO
Thirty-eight patients were treated with high dose rate endobronchial brachytherapy to palliate symptoms (cough, hemoptysis, fever, and/or shortness of breath) caused by endobronchial of previously irradiated (greater than or equal to 5000 cGy) bronchogenic carcinoma. The dose per fraction was 600 cGy at a radius of 1 cm from the center of the linear path of the source, and each patient received three fraction, each fraction separated by a 1-week interval. Twenty-nine patients (76%) had symptomatic improvement, 16 with complete and 13 with partial relief of symptoms. The likelihood of symptom relief was greater in those patients who had extra-bronchial tumor measuring less than 5 cm (15/15) compared to those with extra-bronchial tumor measuring greater than or equal to 5 cm (2/8). The median duration of symptom relief was 7.5 months. Repeat bronchoscopy done 3 months after brachytherapy revealed that 41% (11/27) had complete tumor regression and another 41% (11/27) had partial regression. Nine of 14 patients with post-obstructive atelectasis/pneumonitis had radiographic improvement. Twelve patients (32%) died from massive hemoptysis occurring 2-56 weeks (median 10 weeks) after brachytherapy. Location of the recurrence was the most important predictor of pulmonary hemorrhage. It occurred only in patients with recurrence in the right upper lobe, right mainstem, or left upper lobe bronchus. Whether this high rate of fatal pulmonary hemorrhage was a real phenomenon or a statistical fluke of small numbers remains an unanswered question.
Assuntos
Braquiterapia/métodos , Carcinoma Broncogênico/radioterapia , Neoplasias Pulmonares/radioterapia , Recidiva Local de Neoplasia/radioterapia , Dosagem Radioterapêutica , Braquiterapia/efeitos adversos , Carcinoma Broncogênico/mortalidade , Causas de Morte , Hemorragia/etiologia , Humanos , Pneumopatias/etiologia , Neoplasias Pulmonares/mortalidade , Recidiva Local de Neoplasia/mortalidadeRESUMO
Two tripeptide analogues (N-[3-methyl-1-S[[2-S [(methyl-amino)carbonyl]-1-pyrrolidinyl] carbonyl]butyl-D-analine) (SC40476) and N-[3-methyl-S-(1-pyrrolidinylcarbonyl)butyl]-D-alanine, ethyl ester, hydrochloride (SC42619], inhibit aggregation of, and secretion from, human platelets induced by thrombin but cause no significant inhibition of esterolysis or fibrin formation catalysed by this enzyme. Inhibition by SC40476 of the aggregatory response induced by thrombin is incomplete. Neither peptide analogue inhibits aggregation induced by ADP, collagen, vasopressin or 11,9-epoxymethanoprostaglandin H2 (U-46619). Enhancement of the response is observed when nonsaturating concentrations of these agonists are employed. SC42619 causes a parallel shift to the right in the concentration-response curve describing aggregation induced by thrombin. The Schild plot of these data has a slope of 1.05 and the pA2 is 2.9 +/- 0.1. Both SC40476 and SC42619 induced a small but significant decrease in the single platelet content of platelet suspensions. Neither peptide analogue increases platelet cytosolic [Ca2+] measured using quin 2 or Fura 2. Both analogues cause inhibition of the increase in cytosolic [Ca2+] induced by thrombin. Inhibition by SC42619 is competitive with respect to thrombin when the extracellular [Ca2+] is reduced to less than 0.1 microM but is non-competitive in the presence of 1 mM Ca2+. SC42619 also inhibits the increase in cytosolic [Ca2+]induced by ADP in the presence of 1 mM Ca2+ but not the smaller increase caused by this agonist when the medium contains less than 0.1 microM Ca2+. SC42619 inhibits Mn2+ influx induced by thrombin and ADP. SC40476 and SC42619 inhibit the enhanced incorporation of [32P] into phosphatidic acid observed on stimulation by thrombin of platelets pre-labelled with [32P]-phosphate. Addition of the peptide analogues alone fails to increase significantly the 32P content of phosphatidate, phosphatidylcholine, phosphatidylserine or phosphatidylethanolamine. SC40476 causes no detectable hydrolysis of glycoprotein V as detected by release of the proteolytic product (glycoprotein VFR). The results indicate that SC40476 and SC42619 interact selectively with the platelet thrombin receptor. Both peptide analogues act as effective antagonists for this receptor but also possess weak agonist activity which may also result from interaction with the thrombin receptor. The molecular basis for this latter activity has not been defined. SC42619 non-selectively inhibits Ca2+ influx induced by several agonists but this effect does not appear to contribute to the observed inhibition of the aggregatory and secretory responses.
Assuntos
Antitrombinas/farmacologia , Plaquetas/efeitos dos fármacos , Peptídeos/farmacologia , Receptores de Superfície Celular/efeitos dos fármacos , Plaquetas/metabolismo , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Glicoproteínas/metabolismo , Humanos , Hidrólise , Fosfolipídeos/metabolismo , Receptores de Trombina , Trombina/farmacologiaRESUMO
Twelve patients with incompletely resected meningiomas were treated with postoperative radiation therapy. Nine of these patients had previously undergone incomplete surgical resection, and three had suffered one or more postoperative recurrences. The median dose of irradiation was 5490 rads in 6 weeks (range 4800 to 6080 rads). All patients were followed with serial neurological examinations and computerized tomography (CT) scans. Median follow-up period was 54 1/2 months (range 20 to 120 months); 10 of the 12 patients were followed for longer than 42 months posttreatment. Nine patients had no clinical evidence of recurrent disease after radiation therapy, and CT scans confirmed lack of progression or a gradual decrease in tumor size. Three patients had tumor recurrences; two of these lesions appeared at 70 and 112 months after irradiation as extracranial extensions beyond the margin of the irradiation field, and one has exhibited recurrence within the field at 48 months. Three patients who were treated after prior recurrences have demonstrated prolonged progression-free intervals in comparison to the intervals between recurrences prior to irradiation. No significant complications attributable to treatment have been found in any of the patients. These results are discussed in relation to previous reports of the incidence of meningioma recurrence after incomplete resection.
Assuntos
Neoplasias Meníngeas/radioterapia , Meningioma/radioterapia , Adolescente , Adulto , Idoso , Criança , Feminino , Humanos , Masculino , Neoplasias Meníngeas/diagnóstico por imagem , Neoplasias Meníngeas/cirurgia , Meningioma/diagnóstico por imagem , Meningioma/cirurgia , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/prevenção & controle , Recidiva Local de Neoplasia/radioterapia , Tomografia Computadorizada por Raios XRESUMO
This study evaluated the validity and reliability of a modified qualitative dietary fat index questionnaire (QFQ) in an adolescent minority population. The QFQ was administered to study participants twice over a 2-week period, and data were compared with mean values from three 24-hour recalls. Fifty-seven low-income, overweight, African American adolescent girls, aged 11 to 17 years, were recruited from 7 public housing developments in Atlanta, Georgia. To determine validity, the total QFQ score was compared with the mean values of total fat, percentage of energy from fat, and total energy from three 24-hour recalls within 2 weeks of first administration of the QFQ. Reliability was tested in a subsample (n = 22) by comparing total QFQ scores administered 2 weeks apart. Total fat was significantly correlated (r = 0.31, P < .05) with the QFQ score. Total energy (r = 20.23) and percentage of energy from fat (r = -0.23) were not significantly correlated with the QFQ score. The test-retest QFQ scores were significantly correlated (r = 0.54, P < .01). The data suggest that additional modifications are needed to make the QFQ more appropriate for low-income, over-weight, African American adolescent girls.
Assuntos
Fenômenos Fisiológicos da Nutrição do Adolescente , Negro ou Afro-Americano , Fenômenos Fisiológicos da Nutrição Infantil , Registros de Dieta , Gorduras na Dieta/administração & dosagem , Obesidade , Adolescente , Índice de Massa Corporal , Peso Corporal , Criança , Ingestão de Energia , Feminino , Georgia , Humanos , Grupos Minoritários , Avaliação Nutricional , Obesidade/etnologia , Pobreza , Reprodutibilidade dos Testes , Fatores Socioeconômicos , Inquéritos e QuestionáriosRESUMO
As a pilot experiment towards the reconstruction of human chromosomes from their electron microscopic projections, a chromosome model was photographed and several cross-sectional planes successfully reconstructed. Some practical constraints and conditions for this type of work are defined.
Assuntos
Cromossomos Humanos/ultraestrutura , Modelos Estruturais , Cromatina/ultraestrutura , Computadores , Apresentação de Dados , Humanos , Modelos Moleculares , Tomografia Computadorizada por Raios XRESUMO
The identification of victims of an aircraft accident may be very difficult because of the degree of fragmentation associated with the accident. Periodically, the Divisions of Aerospace Pathology and Toxicology have been asked to identify tissue, bone, or bloodstains of undetermined origin. Usually this request has been precipitated by situations in which a) it is questionable whether an aircraft has sustained a bird strike, b) unidentified pieces of tissue are found floating at sea, or c) fragments of bone, tissue, or blood-stained flight apparel are found near a crash site. Preliminary studies have shown that gross examination and the methods and procedures used in forensic serology may also be applied in aircraft investigation with very good results. These methods are used as an aid to confirm the identity of the victims involved.
Assuntos
Acidentes Aeronáuticos , Medicina Legal , Animais , Manchas de Sangue , Humanos , Técnicas Imunológicas , Testes de Precipitina , Especificidade da Espécie , Manejo de EspécimesAssuntos
Desoxiuridina , Deficiência de Ácido Fólico/diagnóstico , Deficiência de Vitamina B 12/diagnóstico , Anemia Megaloblástica/complicações , Anemia Megaloblástica/diagnóstico , Medula Óssea/metabolismo , DNA/biossíntese , Deficiência de Ácido Fólico/complicações , Humanos , Métodos , Deficiência de Vitamina B 12/complicaçõesAssuntos
Epinefrina/sangue , Fator de Ativação de Plaquetas/análise , Agregação Plaquetária , Serotonina/sangue , Triazóis , Apirase/farmacologia , Azepinas/farmacologia , Contagem de Células/efeitos dos fármacos , Dioxanos/farmacologia , Relação Dose-Resposta a Droga , Hirudinas/farmacologia , Humanos , Idazoxano , Indometacina/farmacologia , Ketanserina/farmacologia , Triazinas/farmacologiaAssuntos
Traumatismos Abdominais , Traumatismos Abdominais/diagnóstico , Traumatismos Abdominais/diagnóstico por imagem , Traumatismos Abdominais/etiologia , Traumatismos Abdominais/cirurgia , Procedimentos Cirúrgicos do Sistema Digestório , Hemorragia/prevenção & controle , Humanos , Rim/cirurgia , Fígado/cirurgia , Métodos , Pâncreas/cirurgia , Radiografia , Fraturas das Costelas/diagnóstico , Ruptura/cirurgia , Baço/cirurgia , Fatores de TempoRESUMO
In aspirin-treated platelets labelled by preincubation with [(3)H]-choline, enhanced release of both [(3)H]-choline and [(3)H]-choline phosphate resulted from stimulation by collagen or thrombin. No such release accompanied stimulation by ADP, platelet-activating factor or adrenaline. Release of [(3)H]-choline phosphate was entirely dependent on aggregate formation whereas release of [(3)H]-choline was reduced but not eliminated, if aggregation was prevented. The properties of [(3)H]-choline and [(3)H]-choline phosphate release indicated that both collagen and thrombin induced activation of phospholipase D in the absence of aggregate formation. Such activation was augmented if aggregate formation occurred. Aggregation induced by these two agonists also caused activation of phosphatidylcholine-specific phospholipase C. These effects were prevented in the presence of staurosporine and could also be induced by addition of a synthetic 1,2-diacylglycerol indicating a role for protein kinase C.
RESUMO
When aggregation is measured as the disappearance of single platelets synergistic interaction between excitatory agonist pairs can be observed using washed platelets in a modified Tyrode's medium or platelet-rich plasma anticoagulated with hirudin; but not using citrated platelet-rich plasma. For aggregation induced by the ADP/adrenaline agonist pair, both the observation of synergistic interaction and the sensitivity of the platelets to these agonists, is a function of extracellular [Ca(2+)]. Synergistic interaction and reduced sensitivity to the individual agonists, especially adrenaline, is observed when extracellular [Ca(2+)] > 100 µM. The data suggest that lower affinity binding of Ca(2+) to the glycoprotein IIb/IIIa complex may modulate platelet sensitivity to these excitatory agonists. The conditions used to resuspend the platelets also influences the nature of the response to the ADP/adrenaline agonist pair and the sensitivity of the platelets to these agonists. A synergistic response and/or reduced sensitivity to ADP is observed on resuspension in modified Tyrode's medium but does not occur on resuspension in citrated plasma or in plasma anticoagulated with hirudin. The factor responsible for enhancing sensitivity, and hence abolishing the synergistic response, is a species of low molecular weight (M(r) less than 25 KDa). It is neither citrate nor Ca(2+).
RESUMO
The biochemical relationship between the red cell antigens Xga and the MIC2 gene product, CD99--previously designated the 12E7 antigen--has been examined by immunoblotting and immunoprecipitation analyses of the protein molecules bearing these antigens. Immunoblotting of membrane components and Xga-immunoprecipitates with anti-Xga has shown that Xga antigen is carried on a broad band of apparent molecular weight (Mr)) 24,500-29,500, which consists of a dark stained component at M(r)24,500 and a more diffusely stained component at approximately M(r) 26,500-29,500. Immunoblotting of membrane components and 12E7-immunoprecipitates with 12E7, and RFB-1 and NaM123 which also recognise CD99, distinguished two bands of M(r) 30,000 and 32,000. A non-radioactive immunoprecipitation technique was employed, which uses chemiluminescence detection of biotin-labelled red cell proteins. The protein of M(r) 32,000, which carries CD99, was identified by this method and the red cell quantitative polymorphism of CD99 was demonstrated. When the Xga protein was precipitated from biotin-labelled red cells, a protein of M(r) 32,000 was coprecipitated. This suggests that the proteins carrying the Xga antigen and CD99 are associated in the membrane.
Assuntos
Antígenos CD/sangue , Moléculas de Adesão Celular/sangue , Eritrócitos/imunologia , Isoantígenos/sangue , Antígeno 12E7 , Sequência de Aminoácidos , Antígenos CD/genética , Moléculas de Adesão Celular/genética , Clonagem Molecular , Código Genético , Humanos , Imunoquímica , Isoantígenos/genética , Homologia de Sequência de AminoácidosRESUMO
The monoclonal antibody-specific immobilization of erythrocyte antigens (MAIEA) technique is an immunoassay devised primarily for locating blood group antigens on specific red-cell membrane proteins. The assay involves the incubation of intact red cells with two antibodies, one human alloantibody, the other a nonhuman antibody, usually a rodent monoclonal antibody, but polyclonal antibodies of rabbit origin have been utilized. For a positive result, both antibodies must bind to the same membrane protein. The red cells are lysed, the membrane solubilized and the trimolecular complex of two antibodies and membrane protein is captured in a well coated with goat antirodent (or rabbit) immunoglobulin. The immobilized complex is then detected by the use of peroxidase-conjugated goat antihuman (or rodent) immunoglobulin. Negative results, due to mutual blocking between the human and animal antibodies when their epitopes are close together on the same molecule, have permitted a degree of localization of epitopes on some proteins. This has been most effective in the mapping of Cromer blood group system antigens on the complement control protein domains of decay-accelerating factor (DAF, CD55), but has also proved informative in the clustering of antigens on the Lutheran and Kell glycoproteins. MAIEA is an effective tool for the identification of antibodies to Knops-system antigens on complement receptor 1 (CR1, CD35) in immunohaematology reference laboratories. These antibodies are clinically unimportant, but must be identified before they can be ignored for transfusion purposes.