RESUMO
BACKGROUND This systematic review aimed first to integrate the current body of knowledge on the demographic, institutional and psychosocial information on sperm donors, and second to provide insight into the actual experiences of men who donate and the attitudes towards potential donation. METHODS Electronic databases (PUBMED, CINAHL, PsycINFO, Embase and Web of Science) were searched with no date restriction using a specific search strategy followed by a snowball strategy. English language peer-reviewed abstracts and full texts were screened for eligibility and the risk of bias was assessed with 15 criteria. Eligibility, quality assessments and data extraction were performed by two independent researchers, resolving disagreement by discussion. RESULTS The initial search retrieved 857 studies and after quality assessment, 29 studies were retained for data extraction. Data from nine countries were obtained. The review synthesis revealed differences and similarities between actual and potential sperm donors on demographic characteristics, financial compensation and attitudes towards anonymity, disclosure and providing information to potential offspring. A number of methodological shortcomings have been identified in the studies investigating sperm donors. CONCLUSIONS Institutional factors (such as recruitment procedures, altruism versus compensation of sperm donors, anonymity versus open-identity donation) and the impact of changing legislation have largely dominated the studies on sperm donation. Furthermore, studies from countries with a bias towards white Western ideology and interpretation were over-represented. This has resulted in a profile of potential and actual sperm donors in terms of demographics, recruitment strategies, motivation for donation and attitudes regarding anonymity, disclosure, recipients and offspring. However, the psychosocial needs and experiences of the donor, and their follow-up and counselling are largely neglected. This review has identified key issues to inform current practice and the development of pathways of care for sperm donors that reflect the multidimensional nature of sperm donation.
Assuntos
Espermatozoides , Doadores de Tecidos/psicologia , Obtenção de Tecidos e Órgãos , Altruísmo , Atitude , Revelação , Humanos , Masculino , Motivação , AutoimagemRESUMO
The amyloid precursor protein (APP) undergoes two consecutive cleavages by different proteases, beta-secretase and gamma-secretase, leading to the release of an amyloidogenic 4 kDa fragment called amyloid beta (Abeta). Combining immunoprecipitation and mass spectrometry, we characterized soluble Abeta in cultured cell media of mouse neuroblastoma N2a cells and double hAPP/hBACE-1 transfected HEK293. The major Abeta isoforms detected were Abeta11-34, Abeta1-34, Abeta11-40 and Abeta1-40. In this study, we demonstrate that overexpression of human beta-secretase (BACE-1) in HEK293 cells resulted in predominant Abeta cleavage at position Glu(11) rather than Asp(1), as well as increased production of Abeta(x)-34, but not Abeta(x)-40. Incubation of cells with a specific gamma-secretase inhibitor suggests that cleavage of APP at Leu(34) could be mediated by gamma-secretase itself or by a gamma-secretase dependent process.
Assuntos
Peptídeos beta-Amiloides/biossíntese , Endopeptidases/fisiologia , Fragmentos de Peptídeos/biossíntese , Secretases da Proteína Precursora do Amiloide , Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/farmacologia , Linhagem Celular , Meios de Cultura/química , Endopeptidases/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/antagonistas & inibidores , Isoformas de Proteínas/análise , Solubilidade , TransfecçãoRESUMO
SKALP/elafin is an epithelial proteinase inhibitor with antimicrobial properties that is not normally expressed in human epidermis, but is induced under inflammatory conditions and in some types of skin cancer. SKALP is a member of the recently described trappin gene family, which encodes a new class of proteins, characterized by a four-disulphide core and a transglutaminase substrate domain. Polyclonal antisera against SKALP have been shown to be useful for monitoring disease activity in psoriasis and tumour differentiation in squamous cell carcinoma. We developed ten different mouse monoclonal antibodies (mAbs) against synthetic peptides corresponding to a hexapeptide epitope in the transglutaminase substrate domain and three mAbs recognizing an epitope in the proteinase-inhibiting domain. The antibodies could be used with high specificity by immunohistochemistry on formalin-fixed tissue, by affinity chromatography, by Western blotting, and by enzyme-linked immunoadsorbent assay (ELISA) for the detection of SKALP/elafin. These antibodies have several advantages over existing polyclonal antisera, such as a defined epitope, the detection of full-length SKALP/elafin and unlimited supply. An antibody against the hexapeptide epitope, which is common to all known human, simian, bovine and swine trappin family members, was used to immunolocalize bovine trappins expressed in trachea, that have recently been discovered. These mAbs will serve as important new tools to measure SKALP/elafin and trappin family members in research and diagnostics.
Assuntos
Anticorpos Monoclonais/biossíntese , Técnicas Imunológicas , Família Multigênica/imunologia , Proteínas/genética , Proteínas/imunologia , Proteínas/fisiologia , Motivos de Aminoácidos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Western Blotting , Ensaio de Imunoadsorção Enzimática/métodos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/imunologia , Proteínas Secretadas Inibidoras de Proteinases , Proteínas/químicaRESUMO
We previously demonstrated that the oral antipsoriatic dimethylfumarate is an inhibitor of cytokine-induced adhesion molecule expression in endothelial HUVEC cells. We now report the inhibitory effect of dimethylfumarate on tumor-necrosis-factor-alpha- or interleukin-1 alpha-induced intercellular adhesion molecule 1 expression in normal human dermal fibroblasts. Western blots of normal human dermal fibroblast cytoplasmic extracts showed that dimethylfumarate has minor effects on the I kappa B alpha, beta and epsilon proteins: their cytokine-induced degradation and resynthesis is only slowed down, an effect most prominently observed for I kappa B beta. No inhibitory effect of dimethylfumarate was observed on cytokine-induced RelA/p65 or c-Rel accumulation in nuclear extracts of cytokine-treated normal human dermal fibroblast cells. In contrast, cytokine-induced nuclear factor kappa B1/p50 nuclear accumulation was specifically inhibited by dimethylfumarate. This inhibitory effect on nuclear factor kappa B1 nuclear localization in normal human dermal fibroblasts proved sufficient to inhibit nuclear factor kappa B1-RelA binding to nuclear factor kappa B consensus oligonucleotides in DNA binding assays. Likewise, cytokine-induced activation of a pNF kappa B::luciferase reporter construct in transiently transfected normal human dermal fibroblasts was inhibited by dimethylfumarate. The observations support a mechanistic model for the oral antipsoriatic dimethylfumarate in which lowering of nuclear factor kappa B1 leads to changes in the nuclear factor kappa B1-RelA nuclear balance and inhibition of cytokine-induced adhesion molecule expression in normal human dermal fibroblasts.
Assuntos
Citocinas/farmacologia , Fármacos Dermatológicos/farmacologia , Fibroblastos/citologia , Fumaratos/farmacologia , NF-kappa B/genética , Pele/citologia , Transporte Ativo do Núcleo Celular , Células Cultivadas , Fumarato de Dimetilo , Fibroblastos/efeitos dos fármacos , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , NF-kappa B/biossíntese , NF-kappa B/fisiologia , Transdução de Sinais/efeitos dos fármacos , Pele/efeitos dos fármacosRESUMO
Hexitol nucleic acid (HNA) is a new steric blocking oligonucleotide, hybridizing sequence selectively with RNA. The biological activity of HNA was evaluated in an in vitro translation arrest system targeting Ha-ras mRNA and in a cellular system targeting intracellular adhesion molecule-1 (ICAM-1) expression. HNA very efficiently and selectively inhibited Ha-ras mRNA translation (IC(50) of 50 nM) when targeted at the translation initiation region. When targeting at the 12th codon region, a gap-mer approach was needed to inhibit mRNA translation. Similarly, HNA inhibited ICAM-1 expression in keratinocytes when targeting at codon sequences. In this test system, HNA is less active but more selective than phosphorothioates, but needs lipofection to become active in keratinocytes. This new steric blocker may be an efficient antisense agent providing that enough material can be brought into cells.
Assuntos
Genes ras/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/efeitos dos fármacos , Oligonucleotídeos/farmacologia , Células Cultivadas , Citocinas/farmacologia , Expressão Gênica/efeitos dos fármacos , Genes ras/genética , Humanos , Molécula 1 de Adesão Intercelular/genética , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Oligonucleotídeos/química , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacosRESUMO
ICAM-1 protein in keratinocytes is thought to contribute to cutaneous inflammatory reactions. Its induction depends-among others-on cytokines such as TNF-alpha, IFN-gamma, IL-1 or on retinoic acid (RA), a key regulator of epidermal homeostasis. We investigated the effect of treatments with TNF-alpha, RA or their combination on ICAM-1 expression on proliferative or differentiating keratinocytes over an 8 day culture period. Basal ICAM-1 levels were undetectable at low (30 microM) and standard (88 microM) Ca2+ and RA alone did not induce ICAM-1. However, at high Ca2+ (1500 microM), ICAM-1 levels were augmented in response to RA-treatment. TNF-alpha induced a transient ICAM-1 increase in NHK, which reached peak-levels 2-4 days post cytokine stimulus. RA potentiated the TNF-alpha-induced ICAM-1 response in all Ca2+-concentrations. This potentiating effect of RA was confirmed at the mRNA level. In summary, our results establish retinoic acid as an enhancer of TNF-alpha-induced ICAM-1 levels in NHK.
Assuntos
Molécula 1 de Adesão Intercelular/biossíntese , Queratinócitos/metabolismo , Ceratolíticos/farmacologia , Tretinoína/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Cálcio/metabolismo , Células Cultivadas , Sinergismo Farmacológico , Humanos , PeleRESUMO
Most studies on the antipsoriatic mode of action of dimethylfumarate focused on its antiproliferative effects in keratinocytes. Because inflammatory skin diseases are associated with an upregulation of endothelial cell adhesion molecules and because the presence of inflammatory cells in dermis and epidermis is considered an important feature in psoriasis, we tested the effect of DMF on cytokine-induced adhesion molecule expression in HUVEC, using in situ ELISA and Northern blotting. Dimethylfumarate inhibited ICAM-1, VCAM-1, and E-selectin expression and reduced adhesion of U937 cells to stimulated HUVEC. Monoethylfumarate and fumaric acid had no effect. Similar inhibitory effects for DMF on VCAM-1 expression were observed after stimulation of HUVEC with LPS, PMA, IL-4, and IL-1 alpha or in combinations with TNF alpha. These data are in agreement with previously reported effects of DMF on intracellular thiol levels and inhibition of NF-kappa B activation. The inhibitory effect on cytokine-induced endothelial adhesion molecule expression may represent another target of dimethylfumarate in psoriasis.
Assuntos
Moléculas de Adesão Celular/biossíntese , Citocinas/farmacologia , Endotélio Vascular/efeitos dos fármacos , Fumaratos/farmacologia , Northern Blotting , Adesão Celular/efeitos dos fármacos , Fumarato de Dimetilo , Selectina E/biossíntese , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Ensaio de Imunoadsorção Enzimática , Fumaratos/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Interleucinas/farmacologia , Lipopolissacarídeos/farmacologia , Psoríase/tratamento farmacológico , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Cordão Umbilical , Molécula 1 de Adesão de Célula Vascular/biossínteseRESUMO
Tau is a neuronal phosphoprotein the expression of which is developmentally regulated. A single tau isoform is expressed in fetal human brain but six isoforms are expressed in adult human brain, with the fetal isoform corresponding to the shortest adult isoform. Phosphorylation is also developmentally regulated, as fetal tau is phosphorylated at more sites than adult tau. In Alzheimer's disease, the six adult tau isoforms become hyperphosphorylated and form the paired helical filament (PHF), the major fibrous component of the neurofibrillary lesions. One way to identify phosphorylated sites in tau is to use antibodies that recognize phosphorylated residues within a specific amino acid sequence. We here characterize the two novel phosphorylation-dependent anti-tau antibodies AT270 and AT180 and identify their epitopes as containing phosphorylated Thr-181 and Thr-231 respectively. With these antibodies we show that these two threonine residues are partially phosphorylated in fetal and adult tau and almost fully phosphorylated in PHF tau. This result contrasts with previous studies of Ser-202 and Ser-396 which are partially phosphorylated in fetal tau, unphosphorylated in adult tau but almost fully phosphorylated in PHF tau.
Assuntos
Doença de Alzheimer/metabolismo , Anticorpos Monoclonais/imunologia , Epitopos/química , Emaranhados Neurofibrilares/química , Proteínas tau/química , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Sítios de Ligação , Encéfalo/crescimento & desenvolvimento , Química Encefálica , Córtex Cerebral/química , Córtex Cerebral/embriologia , Córtex Cerebral/crescimento & desenvolvimento , Epitopos/metabolismo , Humanos , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Fosforilação , Fosfotreonina/análise , Fosfotreonina/metabolismo , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Proteínas tau/imunologia , Proteínas tau/metabolismoRESUMO
The microtubule-associated protein tau is a major cytoskeletal protein involved in the neurofibrillary tangles of Alzheimer's disease. Although tau is predominantly a neuronal protein, it has been demonstrated in glia and other nonneuronal cells. We describe the presence of microtubule-associated protein tau epitopes in various muscle fiber lesions in oculopharyngeal and Becker muscular dystrophy, dermatomyositis, central core disease, neurogenic atrophy, and in the recovery phase of an attack of malignant hyperthermia. Western blot demonstrated a 100- to 110-kd tau-immunoreactive protein probably corresponding to 'big tau' as described in peripheral nerves. Tau immunoreactivity in muscle fiber lesions usually co-localized with tubulin, although electron microscopy failed to show an increase in microtubules. Tau and tubulin reactivity also correlated with the presence of desmin and vimentin epitopes. Possible explanations for the presence of tau are briefly discussed.
Assuntos
Epitopos/análise , Músculos/química , Doenças Musculares , Proteínas tau/análise , Adulto , Idoso , Criança , Pré-Escolar , Desmina/análise , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Músculos/ultraestrutura , Doenças Musculares/patologia , Tubulina (Proteína)/análise , Vimentina/análise , Proteínas tau/imunologiaRESUMO
Alzheimer's disease is a progressive degenerative dementia characterized by the abundant presence of neurofibrillary tangles in neurons. This study was designed to test whether the microtubule-associated protein tau, a major component of neurofibrillary tangles, could be detected in CSF. Additionally, we investigated whether CSF tau levels were abnormal in Alzheimer's disease as compared with a large group of control patients. We developed a sensitive sandwich enzyme-linked immunosorbent assay using AT120, a monoclonal antibody directed to human tau, as a capturing antibody. With this technique, the detection limit for tau was less than 5 pg/ml of CSF. Using AT8, which recognizes abnormally phosphorylated serines 199-202 in tau, the detection limit was below 20 pg/ml of CSF. However, with AT8, we found no immunoreactivity in CSF, suggesting that only a small fraction of CSF tau contains the abnormally phosphorylated AT8 epitope. Our results indicate that CSF tau levels are significantly increased in Alzheimer's disease. Also, CSF tau levels in a large group of patients with a diversity of neurological diseases showed overlap with CSF tau levels in Alzheimer's disease.
Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Proteínas tau/líquido cefalorraquidiano , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais , Western Blotting , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Pessoa de Meia-Idade , Peso Molecular , Valores de ReferênciaRESUMO
Tau is a neuronal phosphoprotein whose expression is developmentally regulated. A single tau isoform is expressed in fetal human brain but six isoforms are expressed in adult brain, with the fetal isoform corresponding to the shortest of the adult isoforms. Phosphorylation of tau is also developmentally regulated, as fetal tau is phosphorylated at more sites than adult tau. In Alzheimer disease, the six adult tau isoforms become abnormally phosphorylated and form the paired helical filament, the major fibrous component of the characteristic neurofibrillary lesions. We show here that Ser-202 (in the numbering of the longest human brain tau isoform) is a phosphorylation site that distinguishes fetal from adult tau and we identify it as one of the abnormal phosphorylation sites in Alzheimer disease. The abnormal phosphorylation of tau at Ser-202 in Alzheimer disease thus recapitulates normal phosphorylation during development.
Assuntos
Envelhecimento/metabolismo , Doença de Alzheimer/metabolismo , Encéfalo/enzimologia , Córtex Cerebral/metabolismo , Proteínas Quinases/metabolismo , Serina , Proteínas tau/metabolismo , Adulto , Sequência de Aminoácidos , Animais , Córtex Cerebral/crescimento & desenvolvimento , Feto , Humanos , Recém-Nascido , Mutagênese Sítio-Dirigida , Fosforilação , Ratos , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Proteínas tau/genéticaRESUMO
Recently, a mitogen activated protein kinase has been implicated in the generation of a phosphorylated paired helical filament (PHF) epitope recognized by the monoclonal antibody AT8. This epitope consists of phosphorylated serines 199 and/or 202 of the human microtubule associated protein tau. Theoretically, aside from abnormal kinase activity, inhibition of phosphatase activity could also be involved in the abnormal phosphorylation status of the microtubule associated protein tau. To investigate this, we incubated LA-N-5 neuroblastoma cells with okadaic acid, a specific inhibitor of phosphatase 2A. We found that incubating neuroblastoma cells with okadaic acid induces the abnormally phosphorylated AT8 epitope. The effect of okadaic acid is time and dose dependent and is reversible. Our findings suggest that phosphatase activity is important in the regulation of the phosphorylation state of tau. Phosphatases may act directly on tau or may influence the activity of mitogen activated protein kinase. Incubation of LA-N-5 neuroblastoma cells with okadaic acid provides a cellular model in which the generation of a well-defined PHF-tau epitope can be investigated.
Assuntos
Éteres Cíclicos/farmacologia , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Proteínas tau/metabolismo , Epitopos , Humanos , Immunoblotting , Neuroblastoma , Ácido Okadáico , Fosforilação , Células Tumorais Cultivadas , Proteínas tau/imunologiaRESUMO
The growth-associated protein B-50 also termed GAP-43, F1, pp46, P-57 and neuromodulin is a nervous tissue-specific protein kinase C (PKC) substrate that is considered to play a major role in neurite formation, regeneration, and neuroplasticity. We describe the isolation of seven mouse monoclonal antibodies (Mabs) directed against B-50. The Mabs are produced against the bovine B-50, selected by ELISA for cross-reactivity with its human counterpart, and evaluated on Western blots in comparison with the well-characterized affinity-purified rabbit polyclonal antibodies to rat-B-50. The Western blots show that the Mabs NM1, NM4, and NM6 recognize specifically the B-50 of bovine, human, and rat brain extract and the purified PKC phosphorylated and unphosphorylated rat B-50 isoforms. The Mabs NM2 and NM3 cross-react with bovine B-50 immunoreactive c-kinase substrate (BICKS), a protein sharing a 17 amino acid sequence homology with B-50. Two Mabs are useful for the detection of B-50 immunoreactivity in formalin-fixed human and rat brain tissues. In human specimen of the hippocampus, a characteristic neuropil distribution of B-50 is detected by the Mabs. In human muscle, Mabs reveal B-50 in nerve bundles and in axons at motor end plates. Thus, these Mabs are useful in investigating the function and localization of the B-50 protein.
Assuntos
Química Encefálica/fisiologia , Glicoproteínas de Membrana/análise , Músculos/metabolismo , Proteínas do Tecido Nervoso/análise , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Proteína GAP-43 , Humanos , Imuno-Histoquímica , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Músculos/imunologia , Proteínas do Tecido Nervoso/imunologia , Ratos , Ratos EndogâmicosRESUMO
Immunoaffinity chromatography with a monoclonal antibody produced against bovine tau protein was used to purify tau proteins from human brain. Fifty grams of brain tissue yielded approximately 2 mg of pure tau proteins. The affinity-purified human tau was used to produce a high-titered rabbit anti-human tau serum. The monoclonal anti-tau antibody and the polyclonal rabbit anti-tau serum were then used to construct a sandwich enzyme-linked immunosorbent assay for detection of human tau proteins, with a sensitivity of 1 ng/ml.
Assuntos
Cromatografia de Afinidade , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas tau/isolamento & purificação , Animais , Anticorpos Monoclonais , Humanos , Soros ImunesRESUMO
We have developed monoclonal antibodies that detect normal microtubule-associated protein-2 (MAP2) epitopes in routinely fixed, paraffin-embedded tissue. The somatodendritic distribution of MAP2 in bovine and human nervous tissue was confirmed with several of these antibodies. Furthermore, some of these antibodies immunohistochemically labeled certain pathological structures in Alzheimer brain, especially neurites in senile plaques. Electron microscopic observations, however, indicate that these MAP2 epitopes are not located in the Alzheimer paired helical filaments themselves, but in amorphous granular structures coexistent with them. While the pathological nature of these structures is undetermined, they may represent artefactual modifications of normal cytoskeletal components.
Assuntos
Doença de Alzheimer/imunologia , Anticorpos Monoclonais/imunologia , Filamentos Intermediários/imunologia , Proteínas Associadas aos Microtúbulos/imunologia , Doença de Alzheimer/patologia , Animais , Bovinos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Humanos , Hibridomas/imunologia , Hibridomas/metabolismo , Camundongos , Microscopia Imunoeletrônica , Inclusão em ParafinaRESUMO
A modified form of the microtubule-associated protein Tau is the major component of the paired helical filaments (PHF) found in Alzheimer's disease. The characterization of these posttranslational Tau modifications is hindered by the lack of sufficient PHF-Tau-specific markers. Here we describe several monoclonal antibodies, prepared by immunization with PHF, two of which showed a selective specificity for PHF-Tau without cross-reactivity with normal Tau. Epitope recognition by these two monoclonals was sensitive to alkaline phosphatase treatment. In Western blotting these monoclonal antibodies reacted specifically with the abnormally phosphorylated epitopes on Alzheimer's disease-associated PHF-Tau. One of the new antibodies can be used for the construction of a sandwich enzyme-linked immunosorbent assay for the specific detection of PHF-Tau without cross-reactivity to normal Tau proteins.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Monoéster Fosfórico Hidrolases/imunologia , Proteínas tau/imunologia , Animais , Western Blotting , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Neurofilamentos/imunologia , Monoéster Fosfórico Hidrolases/metabolismo , FosforilaçãoRESUMO
A monoclonal antibody, termed NFT200, was raised after in vitro immunization with sonicated neurofibrillary tangle (NFT)-enriched fractions prepared from Alzheimer brain. The antigen to which NFT200 is directed was expressed in the paired helical filaments of NFT in sporadic and familial Alzheimer disease (AD), in the straight filaments of NFT in AD, progressive supranuclear palsy and of Pick bodies, and the NFT in several other conditions such as Parkinson-dementia complex of Guam and subacute sclerosing panencephalitis. Granulovacuolar degeneration of AD was also labeled with NFT200. Hirano bodies and amyloid deposits in AD, as well as Lewy bodies of idiopathic Parkinson disease lacked in the antigen. The NFT200-antigen was also expressed as a phosphatase-insensitive antigen in normal neurofilaments found in spinal cord and peripheral nerve axons but was absent from the perikaryal accumulation of neurofilaments induced by aluminum intoxication. Nevertheless, immunoblot studies failed to detect the NFT200 in isolated preparations of the neurofilament proteins, MAP-2, tau, ubiquitin or A4-amyloid peptide. The results indicate that the NFT200 monoclonal antibody is directed against a phosphatase-insensitive epitope of an axonal protein associated with neurofilaments but is labile to isolation and expressed as a stable epitope of a 200 kDa component of NFT.
Assuntos
Doença de Alzheimer/imunologia , Antígenos/análise , Encefalopatias/imunologia , Filamentos Intermediários/imunologia , Emaranhados Neurofibrilares/imunologia , Doença de Alzheimer/patologia , Antígenos/metabolismo , Encefalopatias/patologia , Epitopos , Humanos , Immunoblotting , Técnicas Imunológicas , Microscopia Eletrônica , Emaranhados Neurofibrilares/patologia , Fosforilação , Coloração e RotulagemRESUMO
IgG1-secreting variants have been isolated from three different IgM-secreting hybridomas, in two instances following in vitro immunization. The method used was based on sequential sublining in combination with selection by an IgG1-specific two-site ELISA system employing two different IgG1-specific polyclonal antisera. Idiotypic identity between the IgG1 variants and their respective IgM parent was demonstrated using syngeneic anti-idiotypic antisera. The antigen binding specificity in the IgG1 variants was also conserved. Isolation of naturally occurring IgG1 switch variants from IgM-secreting hybridomas that are produced after in vivo immunization offers a solution to the major disadvantages associated with the generation of IgM hybridomas.
Assuntos
Hibridomas/imunologia , Imunoglobulina G/isolamento & purificação , Imunoglobulina M/imunologia , Doença de Alzheimer/metabolismo , Animais , Anticorpos Anti-Idiotípicos/imunologia , Encéfalo/metabolismo , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas Imunoenzimáticas , Imunoglobulina G/imunologia , Região de Troca de Imunoglobulinas , Camundongos , Camundongos Endogâmicos BALB C , Neurofibrilas/metabolismoRESUMO
A monoclonal antibody (MAb), termed AMC30, was raised after in vitro immunization with sonicated neurofibrillary tangle (NFT)-enriched fractions prepared from Alzheimer's brain. The antigen to which AMC30 is directed was expressed by microglial cells in senile plaques of Alzheimer's disease (AD). Microglia in the parenchyma surrounding brain tumors or infarctions, multinuclear giant cells, perivascular and parenchymal macrophages throughout the brain of AIDS patients were also labeled. Different non-nervous system lesions in which macrophages participate were also stained. Microglial cells in normal areas of the cortex or white matter were not labeled with MAb AMC30. The antigen to which AMC30 is directed was not detected in normal bone marrow, lymph nodes, lung, or spleen monocytes or macrophages. The epitope recognized by MAb AMC30 was present after formalin fixation and paraffin embedding. Our findings suggest that this MAb is directed against an antigen that is specifically expressed in a subpopulation of microglial cells and macrophages reactive to various pathological conditions.
Assuntos
Doença de Alzheimer/patologia , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Córtex Cerebral/patologia , Neurofibrilas/imunologia , Neuroglia/imunologia , Complexo AIDS Demência/patologia , Doença de Alzheimer/complicações , Amiloide/análise , Amiloidose/complicações , Amiloidose/patologia , Animais , Histocitoquímica , Humanos , Hibridomas/imunologia , Técnicas Imunoenzimáticas , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Neurofibrilas/patologia , alfa 1-Antiquimotripsina/análiseRESUMO
Cellular immune reactions against the autoantigen myelin basic protein (MBP) are strongly implicated in the occurrence of postinfectious and postvaccination encephalomyelitis. Clinical autoimmune encephalomyelitis in experimental animals can be transferred with cloned MBP-specific cytolytic major histocompatibility complex Class II-restricted T lymphocytes. The HLA restriction pattern of specific proliferative and cytolytic functions of two human MBP-specific cytotoxic T lymphocyte clones, derived from two different multiple sclerosis patients, was analyzed in detail. Using monoclonal antibodies against various HLA gene products and allogeneic Epstein-Barr virus-transformed B cells as antigen-presenting cells and as targets for cytolysis, it was found that MBP-specific functions of the T cell clones was restricted by HLA class II antigens, and, more specifically, by molecules encoded for by DR locus genes.