RESUMEN
Until today, the oral delivery of peptide drugs is hampered due to their instability in the gastrointestinal tract and low mucosal penetration. To overcome these hurdles, PLA (polylactide acid)-nanoparticles were coated with a cyclic, polyarginine-rich, cell penetrating peptide (cyclic R9-CPP). These surface-modified nanoparticles showed a size and polydispersity index comparable to standard PLA-nanoparticles. The zeta potential showed a significant increase indicating successful CPP-coupling to the surface of the nanoparticles. Cryo-EM micrographs confirmed the appropriate size and morphology of the modified nanoparticles. A high encapsulation efficiency of liraglutide could be achieved. In vitro tests using Caco-2 cells showed high viability indicating the tolerability of this novel formulation. A strongly enhanced mucosal binding and penetration was demonstrated by a Caco-2 binding and uptake assay. In Wistar rats, the novel nanoparticles showed a substantial, 4.5-fold increase in the oral bioavailability of liraglutide revealing great potential for the oral delivery of peptide drugs.
Asunto(s)
Arginina/química , Péptidos de Penetración Celular/química , Liraglutida/administración & dosificación , Liraglutida/efectos adversos , Nanopartículas/química , Polímeros/química , Animales , Células CACO-2 , Supervivencia Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos/métodos , Femenino , Humanos , Inmunoglobulina M , Liraglutida/farmacocinética , Ratas , Ratas Wistar , Técnicas de Síntesis en Fase Sólida , PorcinosRESUMEN
Heat shock proteins such as gp96 (grp94) isolated from tumor or infected cells are able to induce specific cytotoxic T-cell responses and protective immunity. To facilitate rapid and efficient isolation, we generated gp96-specific recombinant single-chain Fv (scFv) antibodies from a semisynthetic phage display library. When immobilized on Sepharose beads, these antibodies allow a high-yield, one-step purification of native gp96 molecules from both mouse and human tumor cell lysates. gp96 molecules eluted from these affinity columns under mild conditions are still capable of generating antigen-specific CTL responses in mice. Thus, scFv-purified gp96 is still associated with peptides; however, in contrast to conventionally purified gp96, scFv-isolated gp96 is free of contaminating material such as mitogenic concanavalin A and proteolytic cathepsins. With the help of these high-yield antibody columns, it is now possible to rapidly isolate immunogenic gp96-peptide complexes from small amounts of tumor material to a purity that allows their use in cancer immunotherapy protocols.
Asunto(s)
Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/aislamiento & purificación , Vacunas contra el Cáncer/aislamiento & purificación , Fragmentos de Inmunoglobulinas/inmunología , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Antígenos de Superficie/inmunología , Antígenos de Superficie/aislamiento & purificación , Vacunas contra el Cáncer/inmunología , Cromatografía en Agarosa/métodos , Proteínas de Choque Térmico/inmunología , Proteínas de Choque Térmico/aislamiento & purificación , Humanos , Fragmentos de Inmunoglobulinas/biosíntesis , Fragmentos de Inmunoglobulinas/genética , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Datos de Secuencia Molecular , Biblioteca de Péptidos , Pruebas de Precipitina , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Homología de Secuencia de Aminoácido , Linfocitos T Citotóxicos/inmunología , Células Tumorales Cultivadas/inmunologíaRESUMEN
In previous studies we described a natural human IgG-anti-F(ab')2 autoantibody family with immunoregulatory properties. Genes coding for the variable regions of the heavy and light chains of the Abs were isolated from a natural Ig gene library and scFv Abs were expressed in E. coli. The scFv Abs bound to F(ab')2 but not to Fab fragments. This points to an epitope located in the hinge region since Fab fragments are lacking most of the hinge. In order to verify our hypothesis, double chain peptides comprising the lower-, middle-, and part of the upper hinge subregion of IgG1-IgG4 were synthesized on cellulose membranes and tested for binding to the Abs. The results show binding of Abs to IgG1 and IgG4 hinge region peptides. In order to identify the key residues of the discontinuous epitopes we carried out complete substitutional analyses in which each amino acid of the wt peptides was substituted by all other amino acids except cysteine. The exchange of proline in the IgG1 or IgG4 middle hinge region abrogated the binding, revealing the importance of this subregion for epitope expression. No binding to the IgG2 or IgG3 hinge was detected. These results indicate that scFv anti-F(ab')2 Abs recognize the hinge region of IgG1 and IgG4 and that the expression of the epitope depends on an intact middle hinge subregion.
Asunto(s)
Anticuerpos Antiidiotipos/metabolismo , Autoantígenos/metabolismo , Sitios de Unión de Anticuerpos , Epítopos/metabolismo , Fragmentos Fab de Inmunoglobulinas/inmunología , Inmunoglobulina G/metabolismo , Fragmentos de Péptidos/inmunología , Secuencia de Aminoácidos , Anticuerpos Antiidiotipos/biosíntesis , Anticuerpos Antiidiotipos/aislamiento & purificación , Humanos , Idiotipos de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/aislamiento & purificación , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismoRESUMEN
We have generated a large complex library of single chain antibodies based on four individual libraries from each of 50 donors. DNA coding for the heavy and light chain variable domains of the IgM and IgG repertoires was amplified by PCR using two different sets of primers. Each individual library was composed of approximately 1-5x10(7) independent clones giving a final combined library of 4x10(9) members. Screening this library by phage display of single chain antibodies with small haptens, peptides and proteins yielded specific antibodies for each class of antigen.
Asunto(s)
Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Biblioteca de Péptidos , Donantes de Sangre , Biblioteca de Genes , Genes de Inmunoglobulinas , Humanos , Inmunoglobulina G/genética , Inmunoglobulina M/genética , Reacción en Cadena de la PolimerasaRESUMEN
The problem of amplifying a specific antibody in a population of millions of other antibodies has been solved by the immune system using the process of clonal selection Binding of an antigen to an IgM receptor on the surface of B-lymphocytes stimulates the proliferation and differentiation of the lymphocyte until it matures to an IgG-producing plasma cell. To mimic the first step of this process in bacteria, vectors have been constructed for the expression of antibodies on the surface of bacteria and phages (for review see Chapter 32 ).
RESUMEN
The application of duplex ultrasonography to the diagnosis of venous thrombosis requires validation by comparison of the duplex findings with the results of ascending contrast venography. In this study, 2534 veins were examined by both methods with contrast venography results serving as the standard for comparison. In this setting, duplex ultrasonography proved to be 100% sensitive and 99% specific for venous thrombosis. Duplex ultrasonography is as reliable as venography in the diagnosis of venous thrombosis and has no associated risks or known complication. In addition, duplex ultrasonography provides information regarding pathologic anatomy that is comparable to the detail provided by high-quality venography. The authors conclude that duplex ultrasonography should be the diagnostic method of choice for evaluating patients with suspected venous thrombosis.
Asunto(s)
Flebografía , Tromboflebitis/diagnóstico por imagen , Medios de Contraste , Extremidades/irrigación sanguínea , Humanos , Sensibilidad y Especificidad , UltrasonografíaRESUMEN
Until now, clinical, noninvasive interrogation of intracranial vessels has consisted only of insonation via transcranial Doppler. Such devices have utilized a 2.0 MHz, continuous wave probe with Doppler spectral waveform display. Clincial aapplication of these techniques has required precise location of cranial "windows" and has been hampered by the extreme anatomic variability of both cranial bony structures and intracerebral arteries. The lack of simultaneous intracranial arterial visualization has limited the clinical pplicability of transcranial Doppler technology. Recently, the authors have utilized a 2.25 MHz curved phased array probe with a pulsed Doppler to image and insonate simultaneously the intracerebral arteries. Colorflow imaging of both near- and far-field regions is the necessary first step for vessel localization and identification. Once this is accomplished, the image of each artery in turn is amplified and gray scale tuning is employed to permit direct visualization of the arterial walls and lumen. Pulsed Doppler waveform analysis is perfomred simultaneously and along the entire visible artery length. In this manner the arteries of both right and left hemispheres are examined in detail. We have found the duplex technique to be superior to the use of Doppler alone in the examination on intracerebral vessels. The ability to visualize and insonate simultaneously eliminates the uncertainty caused by anatomic variation. These advantages, long applied to the evaluation of peripheral vessels, are now available for use in the diagnosis of intracranial arterial disease.