RESUMO
Antibody conjugates were prepared by coupling F(ab')2 or Fab' fragments of an antibody specific for the human high molecular weight-melanoma associated antigen to Fab' fragments of an antibody specific for indium-diethylenetriaminepentaacetate complexes. Monovalent and bivalent haptens were synthesized by reacting the dipeptide tyrosyl-lysine with diethylenetriaminepentaacetic cyclic anhydride. In vitro, the antibody conjugate mediated binding of the 111In-labeled haptens to melanoma cells. In vivo, it allowed specific localization of the haptens in A375 tumors. The bivalent hapten exhibited much higher efficiency at targeting 111In onto cells, both in vitro and in vivo. Antibody conjugate and hapten doses (2 micrograms and 1 pmol, respectively) and the delay between antibody conjugate and tracer injections (24 h) were adjusted to maximize tumor uptake (4% injected dose/g) and tumor to normal tissue contrast (greater than 3) obtained 3 h after injection of the 111In-labeled bivalent hapten. This two-step technique, when compared to direct targeting of 111In-labeled F(ab')2 fragments, provided lower localization of injected activity into the tumor (x 0.25), but higher tumor/tissue ratios, especially with respect to liver (x 7), spleen (x 8), and kidneys (x 10). In addition, high contrast images were obtained within 3 hours, instead of days. Thus, antibody conjugate-mediated targeting of small bivalent haptens, labeled with short half-life isotopes, is proposed as a general method for improving tumor radioimmunolocalization.
Assuntos
Anticorpos/metabolismo , Haptenos/administração & dosagem , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunotoxinas , Radioisótopos de Índio , Melanoma/metabolismo , Proteínas de Neoplasias/imunologia , Ácido Pentético/metabolismo , Animais , Especificidade de Anticorpos , Antígenos de Neoplasias , Feminino , Humanos , Fragmentos Fab das Imunoglobulinas/farmacocinética , Imunotoxinas/metabolismo , Radioisótopos de Índio/imunologia , Melanoma/diagnóstico por imagem , Melanoma/imunologia , Antígenos Específicos de Melanoma , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , CintilografiaRESUMO
A theory of transport via a "carrier" based on Wyman's theory on multiple equilibria is presented. By taking into account the detailed balance principle, it is possible to simplify the flux expressions and their coupling coefficients. In this way, Onsager's rules are derived. An experimental approach to the model is proposed.
Assuntos
Proteínas de Transporte/metabolismo , Modelos Biológicos , Transporte Biológico , TermodinâmicaRESUMO
Early accumulation of maternal mRNA in one of the cells of the cluster of 16 cystocytes is a critical event in the determination of the Drosophila oocyte. A number of developmentally important mRNAs have been shown to accumulate in the early oocyte. We report here the early expression of the yemanuclein-alpha (yem-alpha) transcript, its accumulation in the germarial oocyte and its dynamic localization in the growing oocyte. We have investigated the mechanisms involved in these processes. Microtubules are likely to be involved in both transport and localization as was shown for other maternal transcripts which behave similarly. However, unlike all the cases reported so far, transport and localization are not dependent on 3'UTR sequences. We show that the 5' coding sequence is necessary for the early accumulation of yem-alpha RNA in the oocyte and for its localization pattern during oogenesis.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Drosophila/genética , Proteínas Nucleares/genética , Oócitos/fisiologia , RNA Mensageiro/metabolismo , Animais , Transporte Biológico , Proteínas de Ligação a DNA/metabolismo , Feminino , Microtúbulos/genética , Proteínas Nucleares/metabolismo , Biossíntese de ProteínasRESUMO
The Drosophila yG 4.5 gene (now called yemanuclein-alpha gene), which maps at 98F, is a member of the yema gene cluster isolated in a search for differentially expressed maternal genes. The yemanuclein-alpha transcript (formerly yT 4.5) is specifically expressed in the female germ cells at early oogenic stages and displays a graded distribution along the antero-posterior axis of the oocyte. These provocative features are reminiscent of that of K10, bicoid and Bicaudal-D gene transcripts and lead us to hypothesize that the yemanuclein-alpha gene plays a key role in egg organization. We show in the present work that the yemanuclein-alpha is a nuclear protein highly specific for the oocyte nucleus. The sequence analysis of the 5696 bp EcoRI fragment containing the yemanuclein-alpha gene, and of 5 overlapping cDNAs, reveals a 3006 nucleotides long open reading frame (ORF) flanked by long untranslated 5' and 3' sequences. This ORF predicts a 109,215 kDa protein which is basic (pHi: 8.57), and serine rich (12.08%). It contains a 40 amino acid acidic domain in the first third of the protein with a potential alpha-helix organization; this domain has some similarity with the nucleolin acidic domain. Parts of the yemanuclein-alpha sequence are likely to form secondary structures known to interact with DNA. We demonstrate the DNA binding activity of the yemanuclein-alpha by affinity chromatography experiments. Our data indicate that the yemanuclein-alpha shares some of the features which are characteristic of genuine transcriptional activators.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Proteínas Nucleares/genética , Oócitos/metabolismo , Oogênese , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Núcleo Celular/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Drosophila melanogaster/embriologia , Feminino , Genes , Masculino , Dados de Sequência Molecular , Proteínas Nucleares/fisiologia , Fases de Leitura Aberta , Ovário/metabolismo , Conformação Proteica , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/fisiologiaRESUMO
The different ways of raising antibodies to histamine are reviewed. High affinity monoclonal antibodies could be raised only against derivatized histamine. Succinyl glycinamide derivatization provided the basis of an efficient radioimmunoassay. In this paper the molecular pattern and the thermodynamical properties of histamine recognition were thoroughly investigated. Only the neutral form and not the cationic form of imidazole was recognized. As expected, the ligand recognition increased, with improved structural homology to the immunogen. However, a detailed analysis revealed a zwitterionic effect whenever a carboxylic group was present on the side chain of the ligand.
Assuntos
Anticorpos Monoclonais/imunologia , Haptenos/imunologia , Histamina/análogos & derivados , Histamina/imunologia , Imidazóis/imunologia , Animais , Anticorpos Monoclonais/química , Afinidade de Anticorpos , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Haptenos/química , Histamina/química , Concentração de Íons de Hidrogênio , Cinética , Camundongos , Coelhos , Ratos , TemperaturaRESUMO
Monoclonal antibodies (MAb) to human thyrotropin (hTSH) were prepared by immunization of mice and rats according to different procedures. We have previously demonstrated that a specific antigenic region on the surface of the hTSH molecule was highly immunogenic; in order to produce specific MAb to weakly immunogenic regions of hTSH, we immunized mice and rats with a complex composed of hTSH and an anti-beta hTSH MAb 27 directed against the highly immunogenic region. Monoclonal antibodies elicited by this immunization procedure were highly specific and a high percentage was found complementary to the MAb 27 used in the immunogen. We did not search for anti-MAb 27 antibodies, however one hybridoma produced antibody that preferentially reacted with the immune complex. This antibody, called 515, is an IgG1 that binds the complex with 100-fold greater affinity than it does to the anti-beta hTSH MAb 27 alone. This enhancement was also observed with the Fab fragment of the MAb suggesting that the epitope recognized by this anti-complex MAb is displayed in a very different way when hTSH is bound to the first MAb.
Assuntos
Anticorpos Monoclonais/biossíntese , Complexo Antígeno-Anticorpo/imunologia , Tireotropina/imunologia , Animais , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Especificidade de Anticorpos , Humanos , Imunização , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos EndogâmicosRESUMO
Very sensitive assays of IgE are required for determining prevalence of allergic reactions in children. In order to develop a sensitive two-site IRMA two kinds of mAb were produced. Antibodies specific for D epsilon 1 determinants were derived from immunization with a 40 kDa papain Fc fragment. They bound equally native and 56 degrees C heated IgE. D epsilon 2 specific mAb were obtained after immunization with IgE anti-D epsilon 1 complex and were selected on the basis of their inability to bind heated IgE. In a two-site assay on plastic plates, D epsilon 1 specific mAb led to the binding of IgE but always prevented further binding of anti-D epsilon 1 mAb, anti-human kappa chain mAb or allergen on bound IgE. This was not true when CNBr activated cellulose was used. The influence of the nature of the solid phase disappeared when D epsilon 2 specific mAb were coated on plastic tubes. In this case, the binding of a second mAb with identical or different fine specificity was observed. The best matched pair was E 164 (anti-D epsilon 2) on the solid phase and 6H10 (anti-D epsilon 1) as a tracer. As little as 0.2 UI/ml of IgE could be detected in a 2 hr test.
Assuntos
Anticorpos Monoclonais , Imunoglobulina E/análise , Animais , Especificidade de Anticorpos , Epitopos/imunologia , Feminino , Humanos , Imunoglobulina E/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Plásticos , Conformação Proteica , RadioimunoensaioRESUMO
A novel radioiodinated ligand of the butyrophenone type has been synthesized for the quantification and characterization of dopamine D2 receptors. This haloperidol-derived ligand, haloperidol-succinylglycyl[125I]iodotyrosine ([125I]HSGTI), binds rapidly (equilibrium is reached within 30 min, at 10 pM and 37 degrees C) and with high affinity (Kd = 0.3 nM) to bovine striatal membranes. Its pharmacology, determined by competitive displacement with dopaminergic and non-dopaminergic drugs, is characteristic of binding to dopamine D2 receptors.
Assuntos
Aminoacridinas , Corpo Estriado/metabolismo , Haloperidol/análogos & derivados , Monoiodotirosina/análogos & derivados , Receptores Dopaminérgicos/metabolismo , Animais , Ligação Competitiva , Bovinos , Membrana Celular/metabolismo , Técnicas In Vitro , Radioisótopos do Iodo , Cinética , LigantesRESUMO
Monoclonal antibodies were prepared against pancreatic stone protein, a protein which inhibits calcium carbonate precipitation. Two monoclonal antibodies designated D4 and 2E7 were characterized. Immunoadsorbant columns, obtained by linkage of these monoclonal antibodies to Affigel 10, have been used to isolate immunoreactive forms of pancreatic stone protein from nonactivated human pancreatic juice. These monoclonal antibodies permitted us to test the possible immunological relationship between pancreatic stone protein and human trypsin 1. No immunological similarity was found, in agreement with our previous results, and it was established that pancreatic stone protein is a novel protein and not a degradation product of human trypsin(ogen) 1.
Assuntos
Anticorpos Monoclonais/análise , Proteínas de Ligação ao Cálcio/imunologia , Proteínas do Tecido Nervoso , Tripsina/imunologia , Animais , Ligação Competitiva , Cromatografia em Gel , Humanos , Radioisótopos do Iodo , Litostatina , Camundongos , Camundongos Endogâmicos BALB C , Suco Pancreático/análise , RadioimunoensaioRESUMO
Avidin-coated magnetic beads bind peripheral blood B lymphocytes and monocytes. This unwanted reactivity is not due to the membrane expression of avidin target molecules since beads coated with a biotin-binding analogue are non-reactive and binding occurs even when cellular carbohydrate-binding sites are not active, in the absence of Mg2+ and Ca2+ cations, or when they are blocked by a alpha-D-glucose or alpha-D-mannose in presence of Ca2+ and Mg2+. The non-polar residues of avidin appear not to be engaged in a hydrophobic bond with the membrane molecule since suroptimal quantities of serum albumin do not prevent the avidin binding. It is suggested that ionic interactions explain the binding of avidin-coated beads to B lymphocytes and monocytes and that these can be inhibited with high molecular weight serum molecules or with 0.4 M NaCl.
Assuntos
Avidina/metabolismo , Linfócitos B/metabolismo , Monócitos/metabolismo , Avidina/química , Separação Celular/métodos , Humanos , Técnicas In Vitro , Íons , Magnetismo , Concentração OsmolarRESUMO
We have developed a new protein immunoassay method which uses antibodies raised against synthetic peptides. These synthetic peptides are selected to correspond to fragments of the protein that can be obtained by proteolytic treatment of the protein by trypsin. Just before assay, biological samples are treated with trypsin to liberate the fragments which bind to the anti-peptide antibodies with high affinity. The exact specificity of the assay is predetermined by the amino acid sequence of the fragment which may be either conserved within a family of antigens or, conversely, entirely specific for a particular protein. This method has been successfully employed in the development of an immunoassay for HIV P24 antigen. In that case, peptides were selected that were strongly conserved among the different HIV-1 and HIV-2 strains. This methodology has permitted the development of a sensitive immunoassay with a broad specificity despite many amino acid variations between HIV strains. The methodology could be extended to other protein antigens.
Assuntos
Anticorpos Anti-HIV/imunologia , Proteína do Núcleo p24 do HIV/análise , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Proteína do Núcleo p24 do HIV/imunologia , Imunoensaio/métodos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TripsinaRESUMO
Monoclonal antibodies were prepared against human follitropin by fusion of the myeloma cell line P3-X63-Ag8-653 with spleen cells of mice immunized with either human follitropin or follitropin bound to an anti-beta hFSH monoclonal antibody. The latter immunization procedure permits shielding of the immunodominant specific site and allows the production of numerous specific monoclonal antibodies to human follitropin which are complementary to the monoclonal antibody used in the immunogen. In this investigation two specific monoclonal antibodies were used in a two site immunoradiometric assay which was highly specific, rapid, with one incubation step and demonstrated a sensitivity level of 0.1 mIU/ml. It was possible to differentiate between prepubertal and adult levels of follitropin and to recognize individuals with hyposecretory states.
Assuntos
Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo/imunologia , Hormônio Foliculoestimulante/imunologia , Animais , Feminino , Hormônio Foliculoestimulante/análise , Humanos , Masculino , Camundongos , RadioimunoensaioRESUMO
A new in vitro immunophysical method of removing leukemia or lymphoma cells from autologous bone marrow is described. This new technique makes use of low-density polypropylene beads (density: 0.91) coated with a monoclonal antibody anti-CALLA (antibody ALB2). To ascertain its ability to selectively remove human B/pre-B hematopoietic cells, this technique was applied to normal human bone marrow cell suspensions contaminated with 1-5% of tumor cells. Samples were incubated with the floating beads at 4 degrees C on a rotating wheel for 60 min, followed by a 10-min decantation period, after which the beads bearing the tumor cells floated on the surface, whereas unbound normal marrow cells remained in suspension and were easily recovered free of beads. To demonstrate the feasibility of our method, 2 types of assays were carried out, one using target cell radiolabeled with 111indium, and the other a clonogenic assay. The first assays were to calibrate the different parameters (cellular density, quantity of beads, incubation time) with tumor cell lines: Namalwa (CALLA+) and Molt 4 (CALLA-). These 2 cells lines being able to clone, it is hard to envisage clonogenic assays. In this case, it is very hazardous to evaluate correctly the remaining clonogenic units of Namalwa cells. It is why radiolabelling assays were used for these first experiments. The second assays were to study a model close to the clinical setting and to control the safety of the beads on normal bone marrow cells. In this case, the mixture experiments in which only Namalwa cells were able to clone were evaluated with clonogenic assays, which are more sensitive than radiolabeling assays. A 3- to 4-log reduction of tumor load was achieved with 1-step treatment, and an average of 5-log depletion was obtained by repeating the process twice, as ascertained by the clonogenic assay. Viability, average recovery of nucleated cells, and stem cells potential following the purge were excellent.
Assuntos
Medula Óssea/patologia , Separação Celular/métodos , Leucemia/patologia , Plásticos , Polipropilenos , Anticorpos Monoclonais , Anticorpos Antineoplásicos , Transplante de Medula Óssea , Humanos , Técnicas de Imunoadsorção , Microesferas , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologiaRESUMO
A method of pretargeted immunolocalization of a mouse cell subset, using dual specificity monoclonal antibody conjugates and labeled divalent haptens, is described. Conjugates were prepared by coupling F(ab')2 fragments of an antibody specific for the allelic mouse B cell antigen Lyb8.2, to Fab' fragments of an anti-2,4-dinitrophenyl antibody. Divalent and monovalent haptens were obtained by coupling dinitrophenyl to peptides or to diethylene-triamine-pentaacetic acid. In vitro, divalent haptens bind with higher affinity to mouse spleen lymphocyte-bound than to excess soluble conjugate (affinity enhancement). In vivo, localization of 125I- or 111In-labeled divalent haptens in mouse spleen is much higher than that of the monovalent analogs. Thus, using divalent haptens, a new kind of specificity to target cells was achieved, suggesting that affinity enhancement may improve target to background ratios in radioimmunoscintigraphy.
Assuntos
Anticorpos Monoclonais/imunologia , Haptenos/imunologia , Animais , Especificidade de Anticorpos , Linfócitos B/imunologia , Dinitrobenzenos/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Técnicas In Vitro , Radioisótopos de Índio , Radioisótopos do Iodo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Ácido Pentético/imunologia , Peptídeos/imunologiaRESUMO
Two-step radioimmunotargeting using a bispecific anti-CEA/anti-in-DTPA monoclonal antibody and an 111In-labeled DTPA dimer (diDTPA-TL) was evaluated nine times in eight patients with medullary thyroid cancer (MTC). Immunoscintigraphy was performed 5 and 24 hr after injection of 111In-diDTPA-TL. For five patients, radioimmunoguided surgery (RIGS) was performed using a hand-held gamma probe (sodium iodine), and a biodistribution study was performed 48 hr (four times) and 24 hr (one time) after injection of 111In-diDTPA-TL. Mean tumor uptake (%ID/kg in tumor) was 39 (range 2.75-139). In these five patients, immunoscintigraphy visualized all known tumors and detected unknown foci (US and CT were negative) in the neck (once) and neck and liver (once). Immunoscintigraphy, performed four times in search of a recurrence, detected unknown localizations in the mediastinum and neck (twice) and was negative twice. There were no false-positives. In three of five patients who had surgery, RIGS localized tumor foci not detected by the surgeon. RIGS failed to detect two small lesions (10 x 10 mm) corresponding to sites of fibrosis and microscopic cancer infiltration. Bispecific anti-CEA/anti-In-DTPA mediated targeting of 111In-diDTPA-TL provided elevated tumor uptake and tumor-to-normal tissue ratios. Radioimmunodetection of small MTC lesions is thus possible even when morphological imaging techniques prove negative.
Assuntos
Carcinoma/diagnóstico por imagem , Radioimunodetecção , Neoplasias da Glândula Tireoide/diagnóstico por imagem , Feminino , Humanos , Radioisótopos de Índio , Masculino , Pessoa de Meia-IdadeRESUMO
Eleven patients with primary colorectal carcinoma tumors (4 +/- 2 cm) were given intravenous injections of 1-10 mg of an anti-CEA, anti-In-DTPA bispecific Fab'-Fab monoclonal antibody, and 2-8 days later, were injected with 1.2-4.2 nmol of an 111In-labeled DTPA dimer (6 mCi). The bispecific antibody exhibited good stability and F(ab)'2-like pharmacokinetics. After injection, the 111In-DTPA dimer distributed in a large volume (88 ml/kg-180 ml/kg) and cleared through the kidneys (mean residence time in the whole body: 9 hr-16 hr). Uptake of 111In by the tumor using this two-step technique (1.8%-17.5% injected dose ID/kg, measured from surgical samples 48 hr after hapten injection) was not found significantly lower than that achieved with our reference 111In-labeled anti-CEA F(ab)'2 1 to 4 days after injection in six patients with similar clinical status (5.5%-30.2% ID/kg). In addition, tumor-to-blood and tumor-to-liver uptake ratios were significantly improved (blood 7.8 versus 4.2, liver 2.8 versus 0.8). As a result, low background images allowed detection of 12 of 13 lesions, 4 hr and 24 hr after hapten injection. However, 7 of 11 patients developed HAMA.
Assuntos
Adenocarcinoma/diagnóstico por imagem , Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/imunologia , Neoplasias Colorretais/diagnóstico por imagem , Radioisótopos de Índio , Ácido Pentético , Adenocarcinoma/imunologia , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/imunologia , Feminino , Haptenos , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Radioisótopos de Índio/imunologia , Radioisótopos de Índio/farmacocinética , Masculino , Pessoa de Meia-Idade , Ácido Pentético/farmacocinética , Cintilografia , Sensibilidade e Especificidade , Distribuição TecidualRESUMO
In this study we describe a process for immunopurification of FVIII/vWF complex directly from plasma. A mAb against vWF has been selected that is able to bind, under physiologic conditions, the FVIII/vWF complex and to release it in slightly alkaline conditions while preserving its activity. After investigating the influence of solid supports and of coupling methods on the recovery of active FVIII we produced an immunoadsorbent by immobilisation of the selected mAb onto a Sephacryl S-1000 support using a benzoquinone coupling method. With this immunoadsorbent we developed a purification process directly from plasma with an excellent recovery (50%) of both FVIII and vWF activities. The product obtained is very enriched (the FVIII:C specific activity is 20 IU/mg of protein) and is stable after lyophilization.
Assuntos
Fator VIII/isolamento & purificação , Fator de von Willebrand/isolamento & purificação , Animais , Anticorpos Monoclonais , Soluções Tampão , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Humanos , Concentração de Íons de Hidrogênio , Técnicas de Imunoadsorção , CamundongosRESUMO
A mouse IgG monoclonal antibody (MoAb) directed against the human LFA1 molecule (25.3 MoAb) was used in nine adult leukemic patients to prevent graft rejection after T cell-depleted HLA matched bone marrow transplantation. Based on the results of a previous study in children 0.1 mg/kg of 25.3 was given on days -3, -1, +1, +3, +5 in addition to a standard conditioning regimen with cyclophosphamide (120 mg/kg) and fractionated total body irradiation. The marrow transplant was T cell-depleted using T101 Fab immunotoxin ricin A chain. Seven patients received post-graft immunosuppression with methotrexate and cyclosporine A; two patients received no immunosuppression post-graft. A mean T cell depletion of 98.3% (80-100%) was achieved. Tolerance to the infusions of 25.3 MoAb was excellent. No patient developed any form of graft-versus-host disease. However two patients failed to engraft and three patients had delayed graft failures. These results show that this regimen of anti LFA1 MoAb, which was extremely good at permitting engraftment of HLA mismatched T cell-depleted transplant in children with constitutional diseases, is not able to prevent graft failure and rejection of T cell-depleted HLA matched transplants in adults with leukemia. Further efforts are needed to overcome graft failures in this clinical situation.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos de Diferenciação/imunologia , Transplante de Medula Óssea , Rejeição de Enxerto , Leucemia/cirurgia , Depleção Linfocítica , Linfócitos T/imunologia , Adulto , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Feminino , Teste de Histocompatibilidade , Humanos , Infusões Intravenosas , Leucemia/sangue , Leucemia/complicações , Antígeno-1 Associado à Função Linfocitária , Masculino , Camundongos , Pessoa de Meia-Idade , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/prevenção & controleRESUMO
We report here 16 autologous bone marrow transplantations (ABMT) for poor prognosis B or pre-B malignancies (16 acute lymphoblastic leukemias (ALL), three Burkitt lymphomas, one multiple myeloma) in 11 adults and five children where in vitro purging was accomplished by means of floating immunobeads. This method was developed to avoid non-specific killing by complement or toxin or batch-to-batch variability and provides a 3 log reduction of tumor in a model of B lymphoid malignancies. Low density bone marrow mononuclear cells were incubated for 30 min at 4 degrees C with anti CD10 (ALB2 Immunotech) and/or anti CD19 (Bg4) monoclonal antibodies (MoAb) and then mixed with low density polypropylene beads precoated with a rat antimouse MoAb. After 1 h at 4 degrees C the beads with target cells were decanted; the depleted marrow was collected through a microfilter and cryoperserved. After immunodepletion the recovery of nucleated cells was 75% with a median of 0.75 x 10(8) cells/kg (range 0.3-3.6) and the recovery of hematopoietic progenitors was 83% with a median of 2.9 x 10(4) CFU-GM/kg. The conditioning regimen consisted of busulfan 16 mg/kg and melphalan 140 mg/m2 for three patients, fractionated total body irradiation (TBI) following melphalan 140 mg/m2 for nine patients, TBI and cyclophosphamide 120 mg/m2 for two patients and TBI associated with melphalan and cyclophosphamide for two patients.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Linfócitos B/patologia , Transplante de Medula Óssea , Linfoma de Burkitt/cirurgia , Depleção Linfocítica , Mieloma Múltiplo/cirurgia , Leucemia-Linfoma Linfoblástico de Células Precursoras/cirurgia , Adolescente , Adulto , Anticorpos Monoclonais , Linfócitos B/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Linfoma de Burkitt/epidemiologia , Linfoma de Burkitt/patologia , Criança , Pré-Escolar , Feminino , Seguimentos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Humanos , Masculino , Microesferas , Pessoa de Meia-Idade , Mieloma Múltiplo/epidemiologia , Mieloma Múltiplo/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/epidemiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Prognóstico , Transplante AutólogoRESUMO
We describe a new approach to the detection and quantitation of the enkephalin precursor. The approach is based on the production of antibody against a sequential determinant which is obtained specifically and quantitatively from the enkephalin precursor by tryptic hydrolysis. We chose the hexapeptide Tyr-Gly-Gly-Phe-Met-Arg and developed antibody against the C-terminus of this peptide. The hexapeptide is released from the precursor by mere tryptic cleavage. The antibody permits us to detect the Met-enkephalin precursor at the picomole level. Using this approach, we have quantitated the precursor forms in rat striatum and hypothalamus. The precursor/Met-enkephalin ratio was close to 3/4. This result was in very good agreement with the ratio determined previously in the bovine adrenal medulla.