Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2

The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N- terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.     

The vestibular aqueduct in congenital deafness: evaluation by the axial projection

Thirty-two patients with congenital neurosensory hearing loss demonstrated dilatation of the peripheral limb of the vestibular aqueduct as visualized on pluridirectional tomograms. Multiple additional anomalies involving most frequently the cochlea (15 patients) but also the ossicles (three patients) or vestibule (one patient) were associated. The axial-pyramidal (Poschl) projection was of great value in evaluation of the vestibular aqueduct and cochlea. The technique, clinical correlation, and surgical implications are discussed.     

Smart materials in dentistry

Most dental materials are designed to have a relatively 'neutral' existence in the mouth. It is considered that if they are 'passive' and do not react with the oral environment they will be more stable and have a greater durability. At the same time, it is hoped that our materials will be well accepted and will cause neither harm nor injury. This is an entirely negative approach to material tolerance and biocompatibility and hides the possibility that some positive gains can be achieved by using materials which behave in a more dynamic fashion in the environment in which they are placed. An example of materials which have potential for 'dynamic' behaviour exists with structures which are partly water-based or have phases or zones with significant water content and for which the water within the material can react to changes in the ambient conditions. Such materials may even be said to have the potential for 'smart' behaviour, i.e. they can react to changes in the environment to bring about advantageous changes in properties, either within the material itself or in the material-tooth complex. The controlled movement of water or aqueous media through the material may cause changes in dimensions, may be the carrier for various dissolved species, and may influence the potential for the formation of biofilms at the surface. Some of these issues may be closely interrelated. Clearly, materials which do not have the capacity for water transport or storage do not have the potential for this sort of behaviour. Some materials which are normally resistant to the healthy oral environment can undergo controlled degradation at low pH in order to release ions which may prove beneficial or protective. It is doubtful whether such behaviour should be classified as 'smart' because the material cannot readily return to its original condition when the stimulus is removed. Other materials, such as certain alloys, having no means of transporting water through their structure, can display smart behaviour by undergoing predictable changes in structure in response to applied mechanical or thermal stimuli. It has been difficult to harness such behaviour to the benefit of patients but progress in this area is slowly being made.