Jan A. Hobot

Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry.

We present a simple yet powerful method for the isolation and analysis of exosomes released by antigen-presenting cells (APC). Exosomes are small vesicles (40-90 nm) released by APC, and may have an immuno-regulatory function in vivo. Such exosomes originate from MHC class II peptide loading compartments and, as such, express high levels of MHC Class II. We have utilised magnetic beads, coated with monoclonal antibodies specific for HLA DP, DQ, DR for the specific isolation of exosomes from cell-free supernatants. Beads coated with exosomes are subsequently stained with conjugated antibodies, and analysed by flow cytometry. Characterisation of exosomes by this method demonstrated that exosomes derived from B-lymphocytes express abundant MHC Class I and II molecules. Other immunologically important molecules detected included the co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86). The adhesion molecule ICAM-1 (CD54) was also detected. These exosomes also expressed the B cell marker CD20, and the complement inhibitory protein CD59. The expression of CD63, a lysosomal marker, was variable, and there was no detectable expression of transferrin receptor (CD71). Monocyte derived dendritic cells (cultured for 7 days in GM-CSF/IL-4), demonstrated an immature phenotype, and secreted exosomes with a similar phenotype, with abundant MHC molecules. The expression of CD63 was consistently strong, and the MHC Class I-like molecule CD1a was also present, suggesting a possible function in the presentation of lipid antigens. Again CD59 was expressed suggesting a possible role for APC exosomes in complement regulation. There was no detectable CD71, CD40, CD14, CD20 or CD83. Modification of the extraction protocol allowed a comparative analysis of exosome secretion under various conditions. Treatment of cells with calcium ionophore, or phorbol ester resulted in apparent increases in exosome release, while the phosphatidyl inositol 3-kinase inhibitor, wortmannin, reduced exosome secretion. The immuno-magnetic isolation and analysis of exosomes is a versatile and rapid tool for the analysis of APC exosomes, and may prove a valuable tool for the study of exosome biology.

Scanning electron microscopic examination of bacterial immobilisation in a carboxymethyl cellulose (AQUACEL) and alginate dressings.

Dressings have been applied to open wounds for centuries. Traditionally they have been absorbent, permeable materials, i.e. gauze that could adhere to desiccated wound surfaces, inducing trauma on removal. With the advent of modern wound care products many dressings are now capable of absorbing large volumes of exudate whilst still continuing to provide a moist wound healing environment. Equally important is their ability to lock exudate in the dressing (i.e. bacterial retention within the dressing matrix) such that upon removal from a wound surface bacterial dispersion is minimised. In these studies detailed scanning electron microscopy techniques have demonstrated the fluid controlling properties of alginate wound dressings and a carboxymethylated cellulose wound dressing (AQUACEL) Hydrofiber) dressing (CMCH)). It was demonstrated that following hydration of the latter wound dressing, the subsequent formation of a cohesive gel was effective in encapsulating large populations of potentially pathogenic bacteria such as Psuedomonas aeruginosa and Staphylococcus aureus under the gelled surface, as well as being immobilised within the swollen fibres. In contrast, hydrated alginate wound dressings did not form a uniform, cohesive gel structure, with the result that fewer bacteria were immobilised within the gel matrix. Many bacteria were trapped on individual, non-hydrated fibres. The unique absorbent gelling properties of the CMCH dressing appears to provide an ideal environment for immobilising bacteria.

Resin microscopy and on-section immunocytochemistry /

For the first time, fixation, resin embedding and immunocytochemistry, the subjects of innumerable publications, have been organised into a comprehensive and coherent scheme showing how the various methodologies interrelate. Commercially available resins and their mode of use are described, and the book considers the advantages for cytochemistry and immunocytochemistry of matching tissue fixation to processing and resin embedding. On-section single and double immunolabelling methods are covered, using immunocolloidal gold and immunoperoxidase.

Resins for combined light and electron microscopy: a half century of development.

The last fifty years have seen enormous improvements in the way biological specimens are prepared for microscopy. The Fifties produced the essential groundwork upon which many of our current methodologies are based. Acrylic resin embedding was introduced in 1949, with subsequent publications seeking improvements to resin formulations, embedding protocols, and modes of polymerisation. Procedures for progressive lowering of temperature processing, cryosubstitution, freeze-drying and polymerisation by ultra-violet light at low temperatures, all had their genesis in this decade of great innovation. The Sixties marked the period when the acrylics were eclipsed by the more stable and reliable epoxy resins, and much of our present-day understanding of ultrastructure was elucidated. The Seventies carried on this work with advances in technical developments concerned mainly with freezing methodologies. The beginning of the Eighties saw a resurrection of the acrylic resins, with new formulations of these resins giving reliable and stable embeddings. The low temperature and freezing methodologies pioneered in the Fifties, backed up by recent improvements to low temperature technologies, were used to further our understanding of ultrastructure and breathe new life into the science of immunocytochemistry. The remainder of the Eighties and Nineties has seen the ever increasing application of these various microscopical techniques to a wide range of biological studies. The flexibility offered by the acrylic resins in choosing between different processing, embedding and polymerisation methods has provided the impetus for detailed studies to bring to the attention of microscopists the underlying trends governing specimen preparation. Therefore, looking forward to the new Millennium, this has allowed for a more reasoned choice in organising a strategy to deal with a variety of microscopical requirements and for planning an appropriate protocol.

New aspects of bacterial ultrastructure as revealed by modern acrylics for electron microscopy

Modern acrylics can be used over a wide temperature range (+60 degrees C to -80 degrees C) for infiltration, embedding, and polymerization. They can be used in procedures involving chemical fixation or rapid freezing. This flexibility allows for studies to be carried out upon the effects that different parameters involved in preparing biological tissue for microscopy have upon structure and retention of immunoreactivity. With most preparative methods contributions have been made to our knowledge on bacterial structure in gram-negative and gram-positive cells. The future should lie in integrating the advantages of the various methods for the purpose of advancing our understanding of bacterial structure/function.

Effect of Hydrofiber® wound dressings on bacterial ultrastructure

Ionic silver has well-proven bactericidal properties, and silver-containing wound dressings are now widely used to aid in the creation of an antimicrobial environment in wounds. The effect of silver ions on bacterial ultrastructure can best be studied by viewing bacterial cells under a transmission electron microscope (TEM). Bacterial cells of Pseudomonas aeruginosa were incubated within a control dressing (e.g. a non-antimicrobial Hydrofiber dressing) (Hydrofiber is a registered trademark of E.R. Squibb and Sons, L.L.C.) and a silver-containing Hydrofiber dressing, followed by processing for TEM. Liquid cultures, with and without silver, were prepared for comparison. The addition of silver to growing bacterial cultures stopped growth of the cells very quickly. Ultrastructurally, the presence of silver was found to affect both the shape of the bacterial nucleoid and the organization of bacterial DNA. X-ray microanalysis of bacteria from liquid cultures showed the presence of silver within silver-treated cells and the absence of calcium. It is suggested that the presence of available silver ions within the Hydrofiber dressing could lead to the loss of cellular ions, vital for maintaining the structural integrity of the nuclear area.

Chapter 2 – Bacterial Ultrastructure

Our understanding of bacterial ultrastructure has changed with improvements to light and electron microscopy technologies coupled with the introduction of novel preparative procedures. This chapter, whilst reviewing the variety of structures found in bacteria, also introduces some of the new structural findings that these improvements have revealed and the concepts that they have led to.

2 – Bacterial Ultrastructure

Publisher Summary Bacteria are small, prokaryotic cells, generally of the size of mitochondria. A variety of bacterial shapes can be observed under the light microscope, including cocci, rods, spiral, and even cubes. The inner surface of the cell wall is in contact with a cytoplasmic membrane within which there is the cytoplasm. This contains a nuclear region of DNA, ribosomes and various inclusion bodies. Various surface appendages are present on the outer surface of the bacterial cell wall, which are concerned with the relationship between the bacterium and its external environment. Flagella are the key structures concerned with bacterial motility. Flagella can be located singly at one cell pole (monotrichous flagella), at both poles (amphitrichous flagella), in large numbers along the length of the cell (peritrichous flagella), or as a tuft of flagella at a polar end (lophotrichous flagella). S-layers are found in many types of bacteria, including Gram-positive and Gram-negative bacteria, cyanobacteria, and archaebacteria, and are composed of crystalline arrays of protein or glycoprotein subunits. In Gram-positive organisms, the S-layer lies on the outer surface of the peptidoglycan layer, while in Gram-negative bacteria it is present on the outer surface of the outer membrane.

Resin Embedding and Immunolabelling

The major practical variables involved in resin embedding and immunolabelling have been dealt with in the preceding chapters, but in the discussions of these, other, more theoretical parameters were touched upon. A deeper understanding of such considerations may be important for “fine tuning” the sensitivity that can be obtained when labelling resin sections and for this reason they are more fully examined here.

The Strategic Approach

In biological electron microscopy (EM), the last fifty years have been largely dedicated to the elucidation of cellular ultrastructural detail. The developmental research that has been needed to resolve cell structure is now part of the history of the subject Newman and Hobot 1999). Fixation and resin embedding methods for observing structure have been relegated to the routine in most laboratories. The ultrastructural aim is necessarily towards stabilising tissue to protect its structure during the deleterious process of embedding. Embedding tissue in a powerfully crosslinked resin makes it possible to produce the incredibly thin yet strong sections that resist damage caused by an electron beam and provide high resolution. Since the work of Sabatini, Bensch and Barrnett (1963) double fixation with neutral buffered glutaraldehyde and osmium tetroxide has been confirmed to conveniently preserve the finest ultrastructural detail. Dehydration in an organic solvent gradient is then followed by embedding in an epoxy resin. Araldite for example, is as popular now as it was shortly after the first description of its use by Glauert et al. (1956).