APMIS

The new stereological methods for correct and efficient sampling and sizing of cells and other particles are reviewed. There is a hierarchy of methods starting from the simplest where even the microscopic magnification may be unknown to the most complex where typically both section thickness and the magnification must be known. Opticalsections in suitably modified microscopes can be used to improve the ease and speed with which even the most demanding of these methods are performed. The methods are illustrated by practical examples of applications to a wide range of histological entities including synapses, neurons and cancer cells, glomerular corpuscles and ovarian follicles.

The details of all steps involved in the quantification of biofilm formation in microtiter plates are described. The presented protocol incorporates information on assessment of biofilm production by staphylococci, gained both by direct experience as well as by analysis of methods for assaying biofilm production. The obtained results should simplify quantification of biofilm formation in microtiter plates, and make it more reliable and comparable among different laboratories.

Acute infections caused by pathogenic bacteria have been studied extensively for well over 100 years. These infections killed millions of people in previous centuries, but they have been combated effectively by the development of modern vaccines, antibiotics and infection control measures. Most research into bacterial pathogenesis has focused on acute infections, but these diseases have now been supplemented by a new category of chronic infections caused by bacteria growing in slime‐enclosed aggregates known as biofilms. Biofilm infections, such as pneumonia in cystic fibrosis patients, chronic wounds, chronic otitis media and implant‐ and catheter‐associated infections, affect millions of people in the developed world each year and many deaths occur as a consequence. In general, bacteria have two life forms during growth and proliferation. In one form, the bacteria exist as single, independent cells (planktonic) whereas in the other form, bacteria are organized into sessile aggregates. The latter form is commonly referred to as the biofilm growth phenotype. Acute infections are assumed to involve planktonic bacteria, which are generally treatable with antibiotics, although successful treatment depends on accurate and fast diagnosis. However, in cases where the bacteria succeed in forming a biofilm within the human host, the infection often turns out to be untreatable and will develop into a chronic state. The important hallmarks of chronic biofilm‐based infections are extreme resistance to antibiotics and many other conventional antimicrobial agents, and an extreme capacity for evading the host defences. In this thesis, I will assemble the current knowledge on biofilms with an emphasis on chronic infections, guidelines for diagnosis and treatment of these infections, before relating this to my previous research into the area of biofilms. I will present evidence to support a view that the biofilm lifestyle dominates chronic bacterial infections, where bacterial aggregation is the default mode, and that subsequent biofilm development progresses by adaptation to nutritional and environmental conditions. I will make a series of correlations to highlight the most important aspects of biofilms from my perspective, and to determine what can be deduced from the past decades of biofilm research. I will try to bridgein vitroandin vivoresearch and propose methods for studying biofilms based on this knowledge. I will compare how bacterial biofilms exist in stable ecological habitats and opportunistically in unstable ecological habitats, such as infections. Bacteria have a similar lifestyle (the biofilm) in both habitats, but the fight for survival and supremacy is different. On the basis of this comparison, I will hypothesize how chronic biofilm infections are initiated and how bacteria live together in these infections. Finally, I will discuss different aspects of biofilm infection diagnosis. Hopefully, this survey of current knowledge and my proposed guidelines will provide the basis and inspiration for more research, improved diagnostics, and treatments for well‐known biofilm infections and any that may be identified in the future.

Cytokines represent the major factors involved in the communication between T cells, macrophages and other immune cells in the course of an immune response to antigens and infectious agents. A number of studies on mouse and human T helper (Th) clones have recently provided extensive evidence for the existence of different activities exhibited by Th cells (called Th1 and Th2), which was apparently inferred from the profile of cytokine secretion. The Thl‐type immune response is generally associated with IgG2a production and the development of cellular immunity, the Th2‐type response with IgE production, eosinophils and mast cell production. This review focuses on the role of different cytokines produced by macrophages (especially interferons (IFNs), TNF‐α, IL‐10 and IL‐12) or T cells (IFNs, IL‐2, IL‐4, IL‐10, IL‐13 and TGF‐β) in macrophage‐T cell interactions and the cytokine relevance in the differentiation of Th cells towards the Thl or Th2 type of immune response. Th1‐derived cytokines (IFN‐gamma, IL‐2, TNF‐α) favor macrophage activation, whereas the Th2 cytokines (IL‐4, IL‐10, IL‐13) exhibit suppressive activities on macrophage functions. A key role in the differentiation towards the Th1‐type response is now attributed to IL‐12, a recently described cytokine produced mainly by macrophages. Its production can be upregulated by IFN‐gamma and is inhibited by IL‐10 and IL‐4. All this emphasizes the importance of macrophage‐cytokine interactions in determining the type of immune response. This article also aims to review recent data concerning the roles of IFNs α/β (type I) and IFN‐gamma (type II) in the regulation of the immune response. While there is much information on the regulatory effects of IFN‐gamma (also called “immune IFN”) on the immune response, little is so far known of the role of type I IFNs. These cytokines, originally described as simple antiviral substances, are now taken to be important regulators of the immune response. Recent data indicate that these molecules (especially IFNs‐α) specifically promote the differentiation towards the Th1‐type response. The stimulatory effects of IFN‐α on the generation of the Th1‐type response may be involved in its therapeutic effects in some human diseases, including early AIDS, hypereosinophilia and certain tumors. It is reasonable to assume that, with the increasing interaction between basic and clinical research, considerably more will be understood about how IFNs and other cytokines interact in the modulation of the immune response, and how this knowledge can be successfully translated into new and more selective therapeutic strategies against human diseases.

Lipofuscin (age pigment) is a brown‐yellow, electron‐dense, autofluorescent material that accumulates progressively over time in lysosomes of postmitotic cells, such as neurons and cardiac myocytes. The exact mechanisms behind this accumulation are still unclear. This review outlines the present knowledge of age pigment formation, and considers possible mechanisms responsible for the increase of lipofuscin with age. Numerous studies indicate that the formation of lipofuscin is due to the oxidative alteration of macromolecules by oxygen‐derived free radicals generated in reactions catalyzed by redox‐active iron of low molecular weight. Two principal explanations for the increase of lipofuscin with age have been suggested. The first one is based on the notion that lipofuscin is not totally eliminated (either by degradation or exocytosis) even at young age, and, thus, accumulates in postmitotic cells as a function of time. Since oxidative reactions are obligatory for life, they would act as age‐independent enhancers of lipofuscin accumulation, as well as of many other manifestations of senescence. The second explanation is that the increase of lipofuscin is an effect of aging, caused by an age‐related enhancement of autophagocytosis, a decline in intralysosomal degradation, and/or a decrease in exocytosis.

When the FDA commissioner announced in February 2004 the approval of Avastin for the treatment of patients with colon cancer, he called angiogenesis inhibitors a fourth modality of anti‐cancer therapy. Because angiogenesis inhibitors are relatively less toxic than conventional chemotherapy and have a lower risk of drug resistance, they may also represent a new class of anti‐cancer agents, some of which have sufficiently reduced toxicity that they may be safely used long term. These include immunotherapy, vaccines, telomerase inhibitors, apoptosis inducers, low dose metronomic chemotherapy, novel hormonal therapies, gene therapy and others. However, at least 16 endogenous angiogenesis inhibitors have been discovered in the circulation, and/or in the extracellular matrix. These may become the safest and least toxic of anti‐cancer therapies. Four are already being administered by injection in clinical trials for cancer. Recently, it has been reported that at least two endogenous angiogenesis inhibitors can be significantly increased in humans (endostatin), and in mice (thrombospondin), by oral administration of small molecules which themselves are already FDA approved for other uses. This finding suggests several new clinical applications for the future, including the possibility of guiding the use of angiogenesis inhibitors by blood or urinary biomarkers, currently being developed, that may detect the presence of cancer before it is symptomatic, or before it can be located by conventional methods.

Teeth are colonized by oral bacteria from saliva containing more than 700 different bacterial species. If removed regularly, the dental biofilm mainly comprises oral streptococci and is regarded as resident microflora. But if left undisturbed, a complex biofilm containing up to 100 bacterial species at a site will build up and may eventually cause development of disease. Depending on local ecological factors, the composition of the dental biofilm may vary considerably. With access to excess carbohydrates, the dental biofilm will be dominated by mainly gram‐positive carbohydrate‐fermenting bacteria causing demineralization of teeth, dental caries, which may further lead to inflammation and necrosis in the pulp and periapical region, i.e., pulpitis and periapical periodontitis. In supra‐ and subgingival biofilms, predominantly gram‐negative, anaerobic proteolytic bacteria will colonize and cause gingival inflammation and breakdown of supporting periodontal fibers and bone and ultimately tooth loss, i.e., gingivitis, chronic or aggressive periodontitis, and around dental implants, peri‐implantitis. Furthermore, bacteria from the dental biofilm may spread to other parts of the body by bacteremia and cause systemic disease. Basically, prevention and treatment of dental biofilm infections are achieved by regular personal and professional removal of the dental biofilm.

Lactoferrin is an iron‐binding glycoprotein found in milk, exocrine secretions of mammals, and in secondary granules from polymorphonuclear neutrophils. This review describes the wide spectrum of functions ascribed to lactoferrin, with special emphasis on the antimicrobial properties of this protein, and its derived peptides.