Introduction

Biofilm formation is important in a wide variety of situations: for instance, colonization of pipe surfaces in the food and water industries, metal corrosion due to sulphate reducing bacteria in the shipping and oil industries, and in medicine associated with infections of various tissues (osteomeylitis and endocarditis), dental decay (Addy et al. 1992) and prosthetic implants (Dougherty 1988). Whereas biofilm formation in a chemostat is considered merely an operating nuisance (Bryers 1984), in industrial fermentors such fouling can cause physical damage by the production of metabolites at points on the surface. Biofilms may lead to reduced heat efficiency transfer and reduction in flow rates, and can also act as a resevoir for potential pathogens (Lappin-Scott & Costerton 1989).

Although biofilm formation is frequently associated with being harmful and detrimental, in many instances it can also be beneficial. Biofilms are used in wastewater treatment for the degradation of soluble organic or nitrogenous waste. In nature microbial decomposition of cellulose fibres requires prior attachment of cellulolytic bacteria and Rhizobium cells form biofilms on the roots of leguminous plants where nodules are formed to fix atmospheric nitrogen. Bar-Or (1990) stated the importance of biofilms in stabilizing soil either by acting as cementing agents or flocculating soil particles, thereby improving aeration and water percolation and allowing further microbial growth.

Biofilm formation is difficult to control. A number of authors have reported that biofilm bacteria (sessile) are more resistant to antimicrobial agents than suspended bacteria (planktonic) of the same species (Brown et al. 1988; Anwar et al. 1989). Most commercial biocides and antibiotics were developed and tested for their ability to kill planktonic bacteria (Chopra 1986; Gilbert et al. 1987).

The need for laboratory studies of biofilm communities

If microbial ecology is to move forward, it must go beyond the reduction of the complexities of bacteria into merely isolated cell lines, enzymes, and genetic sequences. A century of pure culture studies has provided extremely detailed information on the biochemistry, physiology and genetics of bacteria. What remains to be determined is how they function as successful members of interacting communities in biofilms and how microbial communities function as components of the environment.

Filling this gap in knowledge involves more than the in situ enumeration of cells, molecules, and genetic sequences. It requires that microbial communities be considered as functional units of ecological activity. Individual microorganisms are often tightly coupled with other community members through a complex network of interactions. The genetic programming of each species may be considered to be a reproductive strategy formulated over 2.5 billion years of natural selection, and intricately intertwined with the survival of other organisms (Margulis 1981). Consequently, the most rigorous measure of understanding must involve not only the cultivation of isolated cell lines, but also the successful cultivation and characterization of dynamic microbial communities, complete with their predators and parasites.

End of the pure culture era

The primary axiom of bacteriology is that organisms must be isolated prior to their identification and study, and prior to the description of new species (Koch 1881, 1884). This axiom is so pervasive that it impacts protozoology, mycology and algology as well as bacteriology.

Introduction

Dental plaque was probably the first biofilm to have been studied in terms of either its microbial composition or its sensitivity to antimicrobial agents. In the seventeenth century, Anton van Leeuwenhoek pioneered the approach of studying biofilms by direct microscopic observation when he reported on the diversity and high numbers of ‘animalcules’ present in scrapings taken from around human teeth. He also conducted early studies on biocides when he established the resistance of these ‘sticky animalcules’ to salt and vinegar.

Following these pioneering observations and until the 1960s, there were relatively few studies of the microbiology of dental plaque. In the past three decades, however, there has been an enormous expansion of knowledge of the biochemistry and bacteriology of the plaque microflora. Impetus for this expansion stemmed from the exploitation of gnotobiotic animal technology in the 1950s and 1960s which established the role of particular bacterial species in the aetiology of two of the commonest diseases to affect humans in industrialised societies, namely, dental caries and periodontal (gum) diseases. The aim of this chapter will be to review our current knowledge of the microbiology of dental plaque, with particular emphasis on properties that relate to its biofilm structure.

Definition of dental plaque

The mouth is unique in the human body in that it provides non-shedding surfaces (teeth) for microbial colonization. Because of this, large masses of bacteria (and their products) are able to accumulate, especially at stagnant sites between teeth (approximal surfaces), in the pits and fissures on occlusal surfaces of premolars and molars, and in the gingival crevice (Fig. 18.1). In contrast, elsewhere in the body, desquamation ensures that the bacterial load is light on mucosal surfaces.

Over the past ten to fifteen years, animal cell biotechnology as a means of producing pure protein products (as opposed to vaccines) has advanced from being an essentially research laboratory-based technology to being a fully fledged player in the biopharmaceutical manufacturing industry. An increasing proportion of products of major clinical importance are produced in animal cells, essentially because accurate and authentic post-translational processing is necessary for the effective functioning of many diagnostic and therapeutic proteins.

This rapid growth to maturity has been made possible by rapid advances in the wide range of technical areas which need to be brought together to achieve safe and cost effective product generation in animal cells. This book has sought to identify these key areas of advancement, to review present progress and to highlight remaining obstacles. The following points represent the major landmarks in the development of animal cells as usable bioreactors and indicate where future research emphasis might be placed to further improve quality and productivity.

The altered regulatory stance to animal cells used as bioreactors

Probably the key event in the resurgence of animal cells as bioreactors for the production of pharmaceuticals was the radical evolution of regulatory attitudes which permitted the use of continuous cell lines as substrates for production. This was made possible by, on the one hand, advances in knowledge of the risks that products from animal cells might pose to patients and a rational assessments of these risks relative to the benefits, and on the other hand, by the development and acceptance of technologies able to reduce these risks to acceptable levels.

Generalities

A bioreactor is essentially a tool or device for generating product using the synthetic or chemical conversion capacity of a biological system. From the 1970s onwards there has been steady increase of attempts to harness biological systems to perform specific and difficult chemical tasks as demands for lower energy consumption and the requirement for ever more intricate syntheses have increased. Bioreactors have ranged from immobilized and engineered enzymes, through an ever increasing number of natural and recombinant microbial systems, to cells derived from animal tissues.

When the aim is to reproduce the complex molecules found in animals, cultured animal cells offer unique qualities as bioreactors because they alone are capable of accurately reproducing effectively the whole of the biological chemistry which operates in our bodies. Thus, they can, in principle, reproduce any molecule occurring there which may find application in medicine. In practice, it is in the production of proteins and their derivatives for diagnostic, prophylactic and therapeutic use that animal cells are currently finding increasing application. The challenge is greatest in the synthesis of significant quantities of human therapeutic proteins intended for clinical approaches that seek to correct deficiencies of endogenous biomolecules or to augment their existing levels. This approach requires high expression levels, stable production capacities, products whose safety and efficacy are rigorously controlled and products of precisely defined structure which accurately reproduce that of the naturally occurring proteins. Despite their unequalled potential to fulfil these criteria, it is only in recent years that animal cells have become useful as practical, industrial-scale bioreactors.

One consequence of more intensive monitoring of the different parameters of cell growth and maintenance in fermentors is that it is becoming possible to understand in finer detail the metabolic requirements of animal cells and how these relate to the efficiency of cells as bioreactors for the production of recombinant proteins and other products. With this understanding it is possible to extend attempts to improve the yield and authenticity of the product to include manipulation of the internal mechanisms of the cell in addition to empirical optimization of the cell's external environment through parameters such as fermentor configuration and medium composition. Specifically, information is accumulating on the effect of cellular metabolism on specific product yield, on the generation of undesirable by-products and on the correctness of post-translational modification. An exciting development is the possibility of specifically tailoring aspects of the cell's metabolism to optimize these functions. Useful results have already been achieved even though the detailed metabolism of cells in culture is still far from being understood.

Energy sources and waste products

Mammalian cells in culture use two main energy sources, glucose and glutamine, for the production of ATP and reduced pyridine nucleotides. The proportion of cellular ATP derived from each substrate varies widely with cell type and culture conditions. However, for most types of cell over half of the ATP generated derives from glutamine oxidation. This can rise to very high levels (>95%) under culture conditions in which lactate production is disfavoured (Reitzer, Wice and Kennell, 1979).

As discussed in Chapter 2, specific protein production (pg/cell/hour) can be dramatically increased by the creation of more efficient producer cells which are capable of high-level transcription coupled with fast and accurate processing and a secretory capacity of matching performance.

However, producing commercial quantities of recombinant protein even by the most productive cells still requires large-scale cell cultures. This need has led to the rapid and continuing development of new approaches to mass cell culture and has been instrumental in dispelling certain dogmas concerning the impracticality of commercial production from animal cells. It has also led to an ongoing re-evaluation of product generation from animal cells according to more sound bio-engineering principles.

Small-scale, low-density animal cell cultures in classical serum-containing medium are relatively simple systems to operate. Since the metabolism of the cells is slow, oxygen demand can be satisfied by simple diffusion or slow stirring, pH changes are slow and easily controlled by buffering and the effect of the production of toxic substances and of proteases are largely abrogated by the absorption and inhibitory capacities of serum.

This situation changes dramatically when large quantities of cells are cultured intensively for the industrial production of kilogram quantities of recombinant proteins and all of these parameters are pushed to new limits. This chapter considers the progress that has been achieved in the key areas to satisfy these demands and discusses the problems that remain and some possible solutions.

All of the procedures and methods discussed so far concern how animal cells can be grown in quantity and engineered to synthesize the maximum possible amounts of protein product required from them. In this section we turn to the methodologies applied to the recovery of the protein product from the cell cultures and to turning it into an acceptable pharmaceutical dosage form. The whole of this operation is known as downstream processing (DSP).

The inclusion of a section on downstream processing in a discussion of animal cells as bioreactors is fully justified for several reasons. First, it is usually the effectiveness of the downstream processing which determines the economic viability of the process since it accounts for at least 70–80% of the overall costs. Second, downstream processing plays a determinant role in assuring the safety of products derived from animal cells. Finally, the success of the overall production process depends to a large extent on the successful integration of the expression system used, the cell culture system used and the processing methods applied to produce the finished product.

Although they may not always be easy to define, interactions exist between all of these stages, and early evaluation of these greatly facilitates successful process design. Too frequently in the past biotechnology companies have developed new cell systems for producing original products and have then adapted standard purification schemes to suit. How ever, there are potentially great savings in time and cost if process design is performed in an integrated manner, for instance, taking into account before the cell culture system is finalized the impact of medium composition or possible cell lysis on the quality of the product stream going for purification.

In the case of virus vaccine production, the actual mass of product required, even in the largest-scale application, is low because a little virus goes a long way. However, when the requirements for therapeutic proteins are considered, a different level of productivity becomes necessary because kilograms or tens of kilograms of pure protein may be needed. To avoid completely impractical culture volumes, the specific production rate of protein by the cells (i.e. picograms of protein per cell per hour) must be increased greatly over that which is normal for animal cells, which, with a few notable exceptions such as gamma globulin secreting cells, globin secreting cells and the cells responsible for the production of some digestive enzymes, do not produce protein at a very high rate.

Two basic approaches can be used to achieve high yields of secreted proteins from animal cells. The first involves tapping the existing potential of some cells to produce and secrete large quantities of a given protein under the control of pre-existing synthetic machinery. This is the situation which exists in antibody-producing cells in which high levels of antibody production are obtained due to the presence of powerful transcriptional enhancers for the immunoglobulin genes. The hybridoma technology proposed by Kohler and Milstein (1975) combines this capacity with the immortality of myeloma cell lines. This technique uses somatic hybridization of primary antibody-producing spleen cells with serially propagated myelomas to permit the long-term production of high levels (up to 500 µg/ml) of the required monoclonal antibody in hybrid cells that can be readily adapted to large-scale culture in fermentors.

General regulatory requirements for biopharmaceuticals

A full discussion of the general regulatory requirements for biopharmaceuticals is beyond the scope of this work but clear guidelines have been laid down in the FDA Points to Consider documents and in the European Community guidelines (Table 6.1), and these documents should be consulted for detailed information on the relevant test programmes.

In general the regulatory bodies call for

• full characterization of the starting materials: gene, genetic construction and host cell.

• full details of the establishment of the banks of production cells, of procedures used for their maintenance and of their stability.

• full details of the production process and the stability of the production cells during the process.

• full details of extraction, purification and characterization of the product.

• evidence of consistency of manufacture.

The guideline documents expand on these requirements and propose techniques that are acceptable and how these should be operated and the results interpreted.

Specific safety issues with animal cells

One of the risks of using cells for the production of biological products is that because they share many of the same biochemical capacities as the patients who will eventually receive the products they are also subject to some of the same pathological events including infection by viruses and the development of oncogenic changes. In principle, the agents causing such events could be transmitted from the production cells to the patients receiving the bioproduct. For this reason, in the early days of the use of cells as substrates for vaccine production, the FDA ruled that only normal cells derived from normal tissues could be used.

The neutrophil, the subject of this book, plays a key role as part of the immune response to microbial infections. Its major function is the rapid killing of bacteria and fungi before they multiply and spread throughout the body. The neutrophil is only one arm of the immune system, which includes other leukocytes, lymphocytes and molecular components such as complement, antibodies, acute phase proteins and cytokines. These cellular and molecular components of immunity constitute a co-ordinated and sophisticated network that has evolved in order to maximise the survival of the host against the range of pathogens it encounters daily. This chapter describes the role of the neutrophil within this immune network. Other elements of the immune system (the cellular and molecular components) are also briefly described but only with emphasis on how they interact with neutrophil function; thus, the descriptions of these systems focus upon how the cellular and molecular elements assist neutrophil function during infection and how neutrophils themselves may affect and regulate other aspects of the immune response. A more complete description of the immune system may be found in texts such as Davey (1989), Roitt (1990) and Benjamini and Leskowitz (1991).

The immune system

The immune system protects humans and animals from microbial infections by such infectious agents as bacteria, yeasts and fungi, viruses and protozoa. These differ greatly not only in their size but in their structural and molecular properties, as well as in the ways in which they seek to infect our bodies. Some of these pathogens infect bodily fluids, some penetrate tissues and some even survive and multiply within individual host cells.

Most if not all of the naturally-occurring neutrophil-activating factors elicit their effects on neutrophils after binding to specific receptors on the plasma membrane. Therefore, this chapter describes how many of these factors are generated (many may be generated by the neutrophils themselves) and how they are thought to mediate their effects; where possible, details of the corresponding receptor will be given. In addition, this chapter includes descriptions of the structure and function of the complement and immunoglobulin receptors involved in the regulation of many neutrophil functions, such as adhesion and opsonophagocytosis.

Leukotriene B4

Properties

Leukotrienes are generated via the activities of lipoxygenases on arachidonic acid. Arachidonic acid itself is generated largely via the activity of phospholipase A2 on membrane polyunsaturated fatty acids, although it may also be formed via the activity of diacylglycerol lipase on sn-l,2-diacylglycerol (see § §6.3.1.1, 6.3.1.5). Different cells possess lipoxygenases that oxidise arachidonic acid at different C atoms on the molecule. For example, platelets possess 12-lipoxygenase, mast cells have 11-lipoxygenase, but neutrophils, eosinophils, basophils, monocytes and macrophages have 5-lipoxygenase. The initial product of 5-lipoxygenase on arachidonic acid is the short-lived molecule 5-hydroxyperoxy-eicosatetraenoic acid (5-HPETE), which is converted into the unstable peroxide LTA4. In neutrophils, monocytes and pulmonary macrophages, this LTA4 is then reduced to LTB4 (Fig. 3.1) via epoxide hydrolase.

Oxygen metabolism and neutrophil function

It has been known for many years that the oxygen metabolism of neutrophils is unusual. As long ago as 1933 Baldridge and Gerard recognised the importance of oxygen for bacterial killing when they observed a ‘respiratory burst’ of increased O2 uptake that accompanied phagocytosis. It was known that, in most cells, O2 was needed to generate energy during mitochondrial respiration, and hence the respiratory burst was mistakenly thought to be required to supply the extra energy needed for the physical processes involved in phagocytosis. It was not until 1959 that the unusual nature, and hence the unusual enzyme system, of the respiratory burst was revealed, when it was shown that phagocytosis and bacterial killing could occur in neutrophils where mitochondrial respiration was poisoned by cyanide. This discovery led to the conclusion that the respiratory-burst enzyme has nothing to do with mitochondrial respiration, and so the extra O2 consumed must serve some other purpose in phagocytosing neutrophils. In fact, mature neutrophils possess very few, if any, active mitochondria, and they obtain their ATP for energy-utilising processes from glycolysis, a process thst does not require O2 supply. This independence of energy generation on O2 supply is important because it means that many neutrophil functions can occur efficiently in the O2-depleted environments that may be found in certain pathological circumstances (e.g. in inflamed tissues). Yet if O2 was not needed by phagocytosing neutrophils for oxidative phosphorylation, then for what other purpose was it required?

At the end of 1980, when I finished writing a book of the cell division cycle with David Lloyd and Robert Poole, I promised myself that I would never write another. During the past few months there have been many times when I wished that I had kept that promise. During this time I have been reassuring my family, colleagues and not least myself, that it was ‘almost finished’. Now that it finally is complete, I promise that I will never write another. Well, perhaps not for a few years yet.

In writing this text, I have been primarily aiming at a level where new researchers to the field can obtain an overview of many of the exciting new developments in neutrophil biochemistry and physiology. To achieve this, I have kept the number of references to original work to a fairly low number. In doing so, I do not wish to detract from the hundreds or, more correctly, thousands of original publications that have arisen over the past 10 or 20 years. Instead, I have tried to make this an ‘easy read’ and so have only quoted key, landmark references or review articles. I hope that most of the important publications and authors are mentioned, and apologise now if I have offended anyone by not directly quoting their articles. To have included all the interesting and relevant publications in this field would have resulted in a text of over twice this size.