J Holland

Protein-loaded poly(dl-lactide-co-glycolide) microparticles for oral administration: formulation, structural and release characteristics

Abstract FITC-labelled bovine serum albumin has been entrapped in sub-5 micron particles of poly( dl -lactide-co-glycolide copolymer) (PLG) using a water-in-oil-in-water (w/o/w) emulsification-solvent evaporation technique. The concentration of PVA stabiliser in the external continuous phase was found to affect not only the particle size, size distribution and protein content but also the release characteristics and internal structure of the microparticles. The importance of primary emulsification was underlined by the finding that the protein content of microparticles with mean size 1 μ m could be increased from about 1% w/w to around 12% w/w by increasing the amount of protein added to the primary emulsion and the homogenisation time in this stage. Under conditions of low stabiliser concentration, multi-nucleate particles formed by polymer precipitation and envelopment of the droplets of the primary w/o emulsion. In this case surface protein loading was of the order of 30% w/w. Under conditions of high PVA stabiliser concentration, disruption of the primary emulsion occurred, resulting in sub-micron particles which were characterised by a high surface protein loading of the order of 70% w/w. A mechanism for protein microencapsulation is presented which is heavily influenced by the shear stresses induced during the process of secondary emulsification. This can explain certain aspects of the relationship between microparticle size and size distribution, protein content and release and the structural characteristics of microparticles produced using the w/o/w emulsification/solvent evaporation technique.

The importance of gastrointestinal uptake of particles in the design of oral delivery systems

Abstract The oral delivery of drugs and vaccines is regarded as the optimal means for achieving therapeutic and prophylactic effects for a number of conditions. For both drug and vaccine delivery the enteric route has the advantages of increased patient compliance, relieves the need for injection and does not require the presence of trained personnel. In the case of vaccination, enteric delivery may result in the induction of a protective mucosal immune response against pathogens which colonise and invade the mucosae. Unfortunately, the oral delivery route is beset with problems such as: gastrointestinal destruction of labile molecules; low levels of macromolecular absorption; and poor immunity usually elicited to orally applied soluble vaccine antigens. To reduce the impact of gut secretions and to ensure the absorption of bioactive agents in an unaltered form, molecules may be incorporated into biodegradable microparticles. This oral delivery system therefore relies on the capacity of the gastrointestinal tract to absorb microparticulate materials, a function which has been demonstrated to be carried out by membranous/microfold (M) cells in the Peyers patches of the mammalian gut. This review examines the nature and extent of particulate absorption by the gut and considers the implications of this process for the oral delivery of drugs and vaccines.

Up-regulation of microsphere transport across the follicle-associated epithelium of Peyer’s patch by exposure to Streptococcus pneumoniae R36a

Transport of antigens through the follicle‐associated epithelium (FAE) of Peyers patch (PP) is the critical first step in the induction of mucosal immune responses. We have previously described that short‐term exposure to Streptococcus pneumoniae R36a induced dramatic morphological alterations of the FAE in rabbit PP. These results prompted us to investigate whether the pneumococci‐induced modifications were accompanied by enhanced ability of the FAE to transport antigens. We addressed this problem by evaluating the ability of the FAE to bind, internalize, and transport fluorescent polystyrene microparticles, highly specific to rabbit M cells, after exposure to S. pneumoniae. Quantitative study revealed a marked increase in the number of microspheres in PP tissues exposed to S. pneumoniae compared to tissues exposed to either phosphate‐buffered saline or Escherichia coli DH5a as controls. No sign of bacterially induced damage to the epithelial barrier was observed. Further confocal microscopy analysis of the FAE surface showed that a significant increase in the number of cells that showed both morphological and functional features of M cells took place within pneumococci‐treated PP tissues. These data provide the first direct evidence that the FAE‐specific antigen sampling function may be manipulated to improve antigen and drug delivery to the intestinal immune system.—Meynell, H. M., Thomas, N. W., James, P. S., Holland, J., Taussig, M. J., Nicoletti, C. Up‐regulation of microspheres transport across the follicle‐associated epithelium of Peyers patch by exposure to Streptococcus pneumoniae R36a. FASEB J. 13, 611–619 (1999)

An experience sampling study of learning, affect, and the demands control support model.

The demands control support model (R. A. Karasek & T. Theorell, 1990) indicates that job control and social support enable workers to engage in problem solving. In turn, problem solving is thought to influence learning and well-being (e.g., anxious affect, activated pleasant affect). Two samples (N = 78, N = 106) provided data up to 4 times per day for up to 5 working days. The extent to which job control was used for problem solving was assessed by measuring the extent to which participants changed aspects of their work activities to solve problems. The extent to which social support was used to solve problems was assessed by measuring the extent to which participants discussed problems to solve problems. Learning mediated the relationship between changing aspects of work activities to solve problems and activated pleasant affect. Learning also mediated the relationship between discussing problems to solve problems and activated pleasant affect. The findings indicated that how individuals use control and support to respond to problem-solving demands is associated with organizational and individual phenomena, such as learning and affective well-being.

The process of individual unlearning: A neglected topic in an under-researched field

In a contemporary business environment where change is often regarded as continuous, the ability of people or organizations to be able to successfully adapt and respond to change is key. Change often involves not only the learning of new behaviours, ideas, or practices but also giving up or abandoning some established ones. Despite both these elements generally being important to change, academic focus on processes of abandoning or giving up established knowledge and practices, that is, unlearning, is lacking. This conceptual article draws on a range of literature to suggest that the process of individual unlearning may have particular features. The review defines the concept of unlearning, differentiates between two different types of individual unlearning, and suggests that each type of individual unlearning may have its own distinctive features and dynamics. This article builds from this insight through developing a typology, which distinguishes between four types of individual unlearning. It concludes with an agenda for future empirical research to examine and validate the concepts presented.

THE IMMUNE RESPONSE TO A MODEL ANTIGEN ASSOCIATED WITH PLG MICROPARTICLES PREPARED USING DIFFERENT SURFACTANTS

The effect of different surfactants on the surface characteristics of poly(D,L-lactideco-glycolide) microparticles prepared by the emulsification/solvent evaporation technique was investigated and the immune response to a protein antigen (OVA) associated with these microparticles was measured. Three surfactants--polyvinyl alcohol (PVA, a conventional stabilizer of PLG microparticles), the non-ionic surfactant, poly(oxyethylene glycerol mono-oleate) [Tagat] and Bile salts (a natural emulsifer)--were used to produce OVA-loaded PLG microparticles. Antigen was detected at the surface of all three types of OVA-loaded microparticles, in amounts in excess of 40% of the total protein load. The levels of specific serum IgG antibody elicited to OVA were significantly higher (P < 0.05) after a single subcutaneous administration of antigen associated with the Bile salts and Tagat formulations compared to the PVA formulation. A strong correlation was revealed between the levels of antibody measured and the magnitude of negative surface charge of the particulate carrier. The pattern of the IgG antibody response to OVA was similar in all three cases, indicating that the degradation rate of the PLG polymer determined the duration of the response. The results demonstrate the potential of using different surfactants to produce PLG microparticles with increased adjuvant activity.

Application of rapid manufacturing techniques in support of maxillofacial treatment: evidence of the requirements of clinical applications

Abstract The concept of applying rapid manufacturing technology to maxillofacial treatment has been described previously; however, these reports did not take into account the practicality of its actual incorporation into clinical practice. Patents in the field are based on imaging techniques combined with rapid manufacturing, which theoretically lead to reconstruction of faces. Some cases studies reported have dealt with the manufacture of prostheses on the laboratory scale. Here two case studies are reported that used imaging and rapid manufacturing techniques for making an ear prosthesis and a burns mask for two patients. Laser scanning was chosen for imaging and Thermojet printing and fused deposition modelling for rapid manufacturing. Outcomes of the study were threefold: improvement in the process, improvement in patient care, and clinical application of existing technology to healthcare. With further research this technology may aid maxillofacial prosthetists in busy facial clinics, reduce patient clinic time, and improve the final product.

Tn916 insertion mutagenesis in Escherichia coli and Haemophilus influenzae type b following conjugative transfer

Transposon Tn916 was shown to be capable of direct conjugative transfer in broth and membrane matings between strains of Escherichia coli K12 and between E. coli K12 and Haemophilus influenzae type b. Only Tn916 was transferred, but Tn916 donor ability was not itself inheritable by the recipients and seemed to be associated with the presence of Tn916 on a non-conjugative pBR322-derived vector in the original donor strain. Transfer of Tn916 by conjugation was found to be an efficient method for producing insertion mutations in the chromosome of recipient cells. Although such insertions were unstable when the cells were grown under non-selective conditions, it was possible to show that over 40% of the isolated Tn916 insertions in the chromosome of E. coli K12 were in gene(s) concerned with histidine biosynthesis, implying that there is a partial hot-spot for Tn916 insertion on the E. coli K12 chromosome. When a strain of H. influenzae type b was used as a recipient, out of approximately 1500 transconjugants tested, two mutants were isolated with insertions in genes controlling the expression of iron-regulated transferrin-binding proteins. These mutants constitutively produced major 76 kDa and minor 90 kDa proteins which bound transferrin, even when grown under iron-sufficient conditions. Tn916 insertion mutagenesis, following transfer by conjugation, is a convenient method for isolating mutations in genes concerned with iron acquisition by this important human pathogen.

Conservation and antigenic cross-reactivity of the transferrin-binding proteins of Haemophilus influenzae, Actinobacillus pleuropneumoniae and Neisseria meningitidis

Haemophilus influenzae acquires iron from the iron-transporting glycoprotein transferrin via a receptor-mediated process. This involves two outer-membrane transferrin-binding proteins (Tbps) termed Tbp1 and Tbp2 which show considerable preference for the human form of transferrin. Since the Tbps are attracting considerable attention as potential vaccine components, we used transferrin affinity chromatography to examine their conservation amongst 28 H. influenzae type b strains belonging to different outer-membrane-protein subtypes as well as six non-typable strains. Whole cells of all type b and non-typable strains examined bound human transferrin; whilst most strains possessed a Tbp1 of approximately 105 kDa, the molecular mass of Tbp2 varied from 79 to 94 kDa. Antisera raised against affinity-purified native H. influenzae Tbp1/Tbp2 receptor complex cross-reacted on Western blots with the respective Tbps of all the Haemophilus strains examined. When used to probe Neisseria meningitidis Tbps, sera from each of four mice immunized with the Haemophilus Tbp1/2 complex recognized the 68 kDa Tbp2 of N. meningitidis strain B16B6 but not the 78 kDa Tbp2 of N. meningitidis strain 70942. Serum from one mouse also reacted weakly with Tbp1 of strain B16B6. Apart from a weak reaction with the Tbp2 of a serotype 5 strain, this mouse antiserum failed to recognize the Tbps of the porcine pathogen A. pleuropneumoniae. However, a monospecific polyclonal antiserum raised against the denatured Tbp2 of Neisseria meningitidis B16B6 recognized the Tbps of all Haemophilus and Actinobacillus strains examined. Since H. influenzae forms part of the natural flora of the upper respiratory tract, human sera were screened for the presence of antibodies to the Tbps. Sera from healthy adults contained antibodies which recognized both Tbp1 and Tbp2 from H. influenzae but not N. meningitidis. Convalescent sera from meningococcal meningitis patients contained antibodies which, on Western blots, recognized the Tbps2s of both pathogens. These data demonstrate the existence of shared epitopes on the Tbps of H. influenzae, N. meningitidis and A. pleuropneumoniae despite their transferrin species specificity.

Application of rapid manufacturing techniques in support of maxillofacial treatment— Evidence of the requirements of clinical applications

The concept of applying rapid manufacturing technology to maxillofacial treatment has been described previously; however, these reports did not take into account the practicality of its actual incorporation into clinical practice. Patents in the field are based on imaging techniques combined with rapid manufacturing, which theoretically lead to reconstruction of faces. Some cases studies reported have dealt with the manufacture of prostheses on the laboratory scale. Here two case studies are reported that used imaging and rapid manufacturing techniques for making an ear prosthesis and a burns mask for two patients. Laser scanning was chosen for imaging and Thermojet printing and fused deposition modelling for rapid manufacturing. Outcomes of the study were threefold: improvement in the process, improvement in patient care, and clinical application of existing technology to healthcare. With further research this technology may aid maxillofacial prosthetists in busy facial clinics, reduce patient clinic time, and improve the final product.