RESUMO
The present report describes the development and application of an efficient method for the direct adsorption/selection of antibody phage using antigens expressed in situ in cryostat tissue sections. In a model system, scFv phage directed towards an epitope on the GA733-2 epithelial glycoprotein expressed in colorectal carcinoma tissue could be specifically enriched up to 1500 fold in single-pass experiments and a million fold after three rounds of selection. Enrichment efficacy was directly proportional to the fraction of antigen positive area over the total area. Sufficient enrichment was achieved at an area fraction of less than four percent, thereby permitting the selection of antibodies to sub-populations of cells or to tissue sub-structures. The general usefulness of the method was demonstrated when a combinatorial scFv antibody phage library derived from melanoma immunized non-human primates was selected in tissue sections of metastatic melanoma. Individual scFv antibodies from enriched phage populations demonstrated different binding specificities, reflected in extracellular and cellular tissue staining patterns which included tumor cell surface reactivity. This method should be particularly useful for the identification of antigens which are only expressed during specific in vivo conditions, and overcomes a major limitation of currently used selection protocols.
Assuntos
Antígenos de Neoplasias/imunologia , Bacteriófago M13/isolamento & purificação , Moléculas de Adesão Celular/imunologia , Fragmentos de Imunoglobulinas/isolamento & purificação , Biblioteca de Peptídeos , Adsorção , Bacteriófago M13/genética , Bacteriófago M13/imunologia , Crioultramicrotomia , Endopeptidases/metabolismo , Molécula de Adesão da Célula Epitelial , Epitopos de Linfócito B/imunologia , Humanos , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/imunologia , Imuno-Histoquímica/métodos , Melanoma/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Células Tumorais CultivadasRESUMO
Belt protected car occupants involved on head-on collisions do not seem to suffer as severe injuries as unembalmed cadavers subjected to comparable simulated head-on collisions. Therefore it has been questioned if cadavers constitute adequate test specimens for study of thoracic tolerances. This investigation compares injuries in safety belt wearing living and dead pigs which have been subjected to simulated head-on collisions on an acceleration tract. Tests were performed on all 20 pigs (10 living and 10 dead). The arterial side of the circulatory system of the dead pigs was infused. The force in the safety belts, the intraaortic pressure, the impact velocity and the deceleration of the sled were recorded. The tests were high speed filmed. Post mortem examination of the pigs revealed differences in injury severity. Dead pigs more easily suffered rib fractures. Deformation of the rib cage due to stripping of the periosteum and laceration of surrounding tissue occurred mainly in the dead pigs. Laceration of intrathoracic blood vessels was seen in dead pigs while isolated heart lesions were seen only in living animals. The main cause of these differences in tolerance level seems to be post mortem changes of the mechanical properties of the different tissues. The results are valid for pigs but indicate that great care has to be exercised when results obtained from cadaver experiments are evaluated concerning thoracic tolerance.
Assuntos
Mudanças Depois da Morte , Cintos de Segurança/efeitos adversos , Aceleração , Acidentes de Trânsito , Adulto , Animais , Condução de Veículo , Cadáver , Desaceleração , Humanos , Suínos , Traumatismos Torácicos/etiologiaRESUMO
Traumatic lesions near the superior margin of the brain frequently are caused by an anterior or posterior blow to the head. The injuries consist of disruptions of one or a few (with subdural bleeding) or of numerous or all (with instant death or immediate unconsciousness and later death because of simultaneous brain stem injuries) parasagittal bridging veins. Cerebral injuries close beneath the superior margin of the brain occur either alone or together with disruptions of parasagittal bridging veins. These injuries consist mainly of subcortical, perivascular or larger, hemorrhages and of injuries to the white matter. No extensive complete necrosis occurs. The lesions are most frequently found in the posterior part of the superior frontal gyrus and the central gyri and often are combined with brain stem injuries. By reconstruction of trauma events the injuries seem to be caused by a rotational acceleration of the head. By model experiments and simulations it is shown that a rotational acceleration of the skull leads to an "inner deformation" of the brain tissue. This deformation may be the explanation of the subcortical injuries, which are therefore designated rotational cerebral injuries (RC-injuries).
