Bacteria in natural, industrial and clinical settings predominantly live in biofilms, i.e., sessile structured microbial communities encased in self‐produced extracellular matrix material. One of the most important characteristics of microbial biofilms is that the resident bacteria display a remarkable increased tolerance toward antimicrobial attack. Biofilms formed by opportunistic pathogenic bacteria are involved in devastating persistent medical device‐associated infections, and chronic infections in individuals who are immune‐compromised or otherwise impaired in the host defense. Because the use of conventional antimicrobial compounds in many cases cannot eradicate biofilms, there is an urgent need to develop alternative measures to combat biofilm infections. The present review is focussed on the important opportunistic pathogen and biofilm model organism Pseudomonas aeruginosa. Initially, biofilm infections where P. aeruginosa plays an important role are described. Subsequently, current insights into the molecular mechanisms involved in P. aeruginosa biofilm formation and the associated antimicrobial tolerance are reviewed. And finally, based on our knowledge about molecular biofilm biology, a number of therapeutic strategies for combat of P. aeruginosa biofilm infections are presented.
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 bridge in vitro and in vivo research 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.
To study type 1 diabetes (T1D), excellent animal models exist, both spontaneously diabetic and virus-induced. Based on knowledge from these, this review focuses on the environmental factors leading to T1D, concentrated into four areas which are: (1) The thymus-dependent immune system: T1D is a T cell driven disease and the beta cells are destroyed in an inflammatory insulitis process. Autoimmunity is breakdown of self-tolerance and the balance between regulator T cells and aggressive effector T cells is disturbed. Inhibition of the T cells (by e.g. anti-CD3 antibody or cyclosporine) will stop the T1D process, even if initiated by virus. Theoretically, the risk from immunotherapy elicits a higher frequency of malignancy. (2) The activity of the beta cells: Resting beta cells display less antigenicity and are less sensitive to immune destruction. Beta-cell rest can be induced by giving insulin externally in metabolic doses or by administering potassium-channel openers. Both procedures prevent T1D in animal models, whereas no good human data exist due to the risk of hypoglycemia. (3) NKT cells: According to the hygiene hypothesis, stimulation of NKT cells by non-pathogen microbes gives rise to less T cell reaction and less autoimmunity. Glycolipids presented by CD1 molecules are central in this stimulation. (4) Importance of the intestine and gliadin intake: Gluten-free diet dramatically inhibits T1D in animal models, and epidemiological data are supportive of such an effect in humans. The mechanisms include less subclinical intestinal inflammation and permeability, and changed composition of bacterial flora, which can also be obtained by intake of probiotics. Gluten-free diet is difficult to implement, and short-term intake has no effect. Regarding the onset of the T1D disease process, slow-acting enterovirus and gliadin deposits are speculated to be etiological in genetically susceptible individuals, followed by the mentioned four pathogenetic factors acting in concert. Neutralization of any one of these factors is capable of stopping T1D development, as lessons are learned from the animal models.
As food safety management further develops, microbiological testing will continue to play an important role in assessing whether Food Safety Objectives are achieved. However, traditional microbiological culture-based methods are limited, particularly in their ability to provide timely data. The present review discusses the reasons for the increasing interest in rapid methods; current developments in the field, the research needs, and the future trends. The advent of biotechnology has introduced new technologies that led to the emergence of rapid diagnostic methods and altered food testing practices. Rapid methods are comprised of many different detection technologies, including specialized enzyme substrates, antibodies and DNA, ranging from simple differential plating media to the use of sophisticated instruments. The use of non-invasive sampling techniques for live animals especially came into focus with the 1990s outbreak of bovine spongiform encephalopathy that was linked to the human outbreak of Creutzfeldt Jakob's Disease. Serology is still an important tool in preventing foodborne pathogens to enter the human food supply through meat and milk from animals. One of the primary uses of rapid methods is for fast screening of large number of samples, where most of them are expected to be test-negative, leading to faster product release for sale. This has been the main strength of rapid methods such as real-time Polymerase Chain Reaction (PCR). Enrichment PCR, where a primary culture broth is tested in PCR, is the most common approach in rapid testing. Recent reports show that it is possible both to enrich a sample and enumerate by pathogen-specific real-time PCR, if the enrichment time is short. This can be especially useful in situations where food producers ask for the level of pathogen in a contaminated product. Another key issue is automation, where the key drivers are miniaturization and multiple testing, which mean that not only one instrument is flexible enough to test for many pathogens but also many pathogens can be detected with one test. The review is mainly based on the author's scientific work that has contributed with the following new developments to this field: (i) serologic tests for large-scale screening, surveillance, or eradication programs, (ii) same-day detection of Salmonella that otherwise was considered as difficult to achieve, (iii) pathogen enumeration following a short log-phase enrichment, (iv) detection of foodborne pathogens in air samples, and finally (v) biotracing of pathogens based on mathematical modeling, even in the absence of isolate. Rapid methods are discussed in a broad global health perspective, international food supply, and for improvement of quantitative microbial risk assessments. The need for quantitative sample preparation techniques, culture-independent, metagenomic-based detection, online monitoring, a global validation infrastructure has been emphasized. The cost and ease of use of rapid assays remain challenging obstacles to surmount.
The intensive research of recent years has suggested that the cause of ulcerative colitis (UC) involves a genetic predisposition to an uncontrolled or unbalanced immune response to luminal or epithelial antigens or against other external factors. Intercellular adhesion molecule-1 (ICAM-1) is pivotal for the influx of neutrophil granulocytes into colonic mucosa, and gene analyses have found polymorphisms in the gene encoding ICAM-1, indicating that changes in ICAM-1 function may be involved in the pathogenesis of UC. Clinical trials of the ICAM-1 antisense oligonucleotide Alicaforsen, which inhibits the synthesis of ICAM-1, have shown positive results in the treatment of patients with left-sided (distal) UC. In addition to emphasizing the central role of ICAM-1 in active stages of UC, the results provide hope for the development and introduction of a more specific and efficient treatment for UC than those currently available. This review discusses the results from studies on the expression of ICAM-1 in colonic tissue from patients with UC.
For many years, attention has been given to the oral bacterium Aggregatibacter actinomycetemcomitans, as a species possibly implicated in the etiology of aggressive periodontitis in adolescents. One of the major virulence factors of A. actinomycetemcomitans is the leukotoxin which is able to kill important cells of the immune system. As demonstrated in population genetic analyses, the population structure of A. actinomycetemcomitans is mainly clonal with evolutionary lineages corresponding to the serotypes. A particular highly leukotoxic clone (JP2) of serotype b has been discovered. The JP2 clone, with an estimated origin some 2400 years ago, is found to be highly conserved, based on analyses of a collection of JP2 clone strains collected through more than 20 years from individuals of diverse origin and living geographically widespread. Despite demonstration of minor evolutionary changes within the genome of JP2 clone strains of A. actinomycetemcomitans, the JP2 clone strains constitute a unique clonal type, the characteristics of which include a 530 basepair deletion in the leukotoxin operon implicated in the enhanced leukotoxic activity of the clone. Mapping of the geographic occurrence of the JP2 clone of A. actinomycetemcomitans has revealed that its colonization is largely restricted to individuals of African descent. Characteristic mutations, which allow JP2 clone isolates from the Mediterranean region to be distinguished from isolates from West Africa, including the Cape Verde islands, suggest that the JP2 clone initially emerged as a distinct genotype in the Mediterranean region of Africa and subsequently spread to West Africa, from where it might have been transferred to the American continent during the transatlantic slave trade. The finding of a sustained selective colonization of individuals of African descent, despite geographical separation from the African continent for centuries, suggests that the JP2 clone might have a distinct host tropism. Further studies are needed to elucidate the reasons for the apparent selective colonization of the Mediterranean and Western African populations. The JP2 clone of A. actinomycetemcomitans appears to play a prominent role in the etiology of aggressive periodontitis compared to other clonal types of the species. While A. actinomycetemcomitans, in general, is considered an opportunistic pathogen of the resident oral microbiota, the JP2 clone has features similar to those of an exogenous pathogen. Clonal types other than JP2 can be isolated from healthy as well as periodontally diseased individuals, whereas the JP2 clone has been isolated primarily from periodontally diseased individuals. As demonstrated in a prospective cohort study in Morocco, where the JP2 clone is endemically present, the presence of this clone in dental plaque confers a remarkably increased risk for development of aggressive periodontitis, suggesting that the JP2 clone is an important etiological agent of aggressive periodontitis in adolescents. Support for association of clonal types other than JP2 of A. actinomycetemcomitans with aggressive periodontitis has also been provided, but the association is much weaker. Nearly half of the JP2 clone carriers were found to be persistently infected during a two-year follow-up period, which indicates a level of stability of colonization with the JP2 clone similar to that previously reported for non-JP2 clonal types of A. actinomycetemcomitans. The relative risk of aggressive periodontitis is highest for individuals with stable JP2 clone colonization. Although the method used is not quantitative, this finding adds to the evidence for a causal role of the JP2 clone in aggressive periodontitis. Longitudinal data shows that few individuals are colonized with the JP2 clone de novo after puberty. Patterns of parent-child carriage and shared colonization of JP2 clone strains among siblings have been demonstrated in other studies, altogether indicating that transmission of the JP2 clone, like other clonal types of A. actinomycetemcomitans, occurs vertically, and through close person to person contacts. In conclusion, a conserved and highly leukotoxic clone of A. actinomycetemcomitans with unique characteristics and with an apparent linkage to individuals of African descent has been identified. Being aware that the genetic constitution of the hosts has not been considered and that focus in our studies has been on A. actinomycetemcomitans only, and not on other members of the oral microbiota, we conclude that the JP2 clone of A. actinomycetemcomitans is a likely etiological agent of aggressive periodontitis in adolescents.
Proteinase 3 (PR3)-anti-neutrophil cytoplasmic antibodies (ANCA) and myeloperoxidase (MPO)-ANCA are suggested to play a pathogenic role as they are closely related to the small-vessel vasculitis syndromes, Wegener's granulomatosis and microscopic polyangiitis. A large body of in vitro and animal experiments supports this concept. The mechanisms of action involve a direct interaction between ANCA and its antigen. The epitope specificity of ANCA may therefore influence the functional effects of ANCA and/or may reflect the mechanisms behind different disease manifestations or disease courses.
The pathogenesis of antineutrophil cytoplasmic antibody (ANCA)‐associated vasculitis (AAV) is unknown but is most consistent with a primary role for neutrophils in the acute injury. Thus, neutrophils are cardinal cells in the pathophysiological process in AAV because they are both effector cells responsible for endothelial damage and targets of autoimmunity. In addition, because of their capacity to synthesize a wide variety of cytokines and chemokines, neutrophils can be considered as important modulators of the inflammatory and potentially of the autoimmune process. ANCA directed against two main autoantigens, namely proteinase 3 and myeloperoxidase, are likely to play a modulatory role in the inflammatory process. Interestingly, neutrophils are an important source of lymphocyte stimulator (BLy), a cytokine that plays a fundamental role in B‐cell physiology, including differentiation, proliferation and immunoglobulin production. The issue of B‐cell activation and/or dysregulation in vasculitis will be discussed.
Granuloma formation is a key pathologic finding in two of the anti‐neutrophil cytoplasmic autoantibody (ANCA)‐associated vasculitides: Wegener's granulomatosis (WG) and Churg–Strauss syndrome (CSS). So far, no animal models have been established convincingly reproducing both vasculitic and granulomatous features typical of WG and CSS. In biopsies, granulomatous lesions are found both at distant extravascular sites and in the vicinity of inflamed vessels, e.g. in the lung. Intriguingly, WG‐granulomata appear to display features of tertiary lymphoid tissue. Cartilaginous and osseous destruction is caused by granulomatous inflammation invading adjacent tissues. Rhinosinusitis is regularly encountered in WG and CSS. Septal perforation, saddle nose deformity, middle and inner ear symptoms, and granulomatous invasion of the palate, orbita, meninges, or the pituitary gland may complicate WG. Both common (e.g. FCGR3B copy number) and distinct (e.g. HLA‐DP, IL‐10.2 ) genetic factors have been identified in AAV potentially favouring inflammation and autoimmunity. The HLA‐DPB1 / RING1 / RXRB region constitutes a quantitative trait locus for ANCA‐positive WG with the strongest association to be reported up to now. A profound alteration of the T‐cell response including Th1 and Th17 responses, anomalously NK‐receptor‐expressing ‘NK‐like’ T cells, and dysfunctional regulatory T cells could facilitate and sustain granuloma formation and autoimmunity.
Because of their multiple overlapping clinical characteristics, Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA) have increasingly been conceptualized as different expressions of a unique anti‐neutrophil cytoplasm antibodies (ANCA)‐associated vasculitis (AAV) disease spectrum. However, this continuum theory remains hindered by uncertainty surrounding a potentially common etiology. This review sheds light on our current understanding of the epidemiology of WG and MPA with the aim of weighing the evidence supporting whether or not these two vasculitis forms are distinct diseases. At present, some epidemiological evidence exists that WG and MPA might correspond to mere variants of a single AAV entity.
Vasculitides are a group of conditions with marked inflammation in and around vessel walls and vascular leakage. These conditions may involve the presence of auto-antibodies such as ANCA or may be mediated by other autoimmune or pathogenic mechanisms. Regardless of the primary trigger, vasculitides entail activation of the complement system as well as the contact/kinin system. In vivo and in vitro data support the involvement of these systems showing activation of the alternative, classical and lectin complement pathways as well as release of bradykinin at sites of vascular inflammation. This short review will summarize some of the data regarding the participation of these systems and the interplay between the complement and kinin systems as well as their interaction with the endothelium and neutrophils. Although these systems do not play a primary role in induction of vasculitis, the peptides released contribute to inflammation and vascular leakage and may thus be identified as potential therapeutic targets.
Antineutrophil cytoplasmic antibodies (ANCA) levels have been thought to follow disease activity, with levels being high at presentation, declining with treatment and increasing just before relapse. However, we have shown that ANCA often persist for many years in patients with clinically inactive Wegener's granulomatosis. ANCA assays are less sensitive for treated disease than for active disease, and the levels in treated patients produce different results in different assay systems. ANCA often persist for years without relapse, and the risk of relapse probably depend on levels that are critical for any individual patient. The capture enzyme‐linked immunosorbent assays may be more sensitive in detecting early relapse. Relapse is more common when ANCA levels are high but, although elevated, ANCA levels are lower in relapse than at presentation. Standardized ANCA levels for the definitions of remission and relapse may not be possible, and the optimal ANCA testing protocol for treated disease remains unclear.
During the first international workshop on antineutrophil cytoplasmic antibodies (ANCA), Copenhagen 25 and 26 January 1988, ANCA, as detected by the indirect immunofluorescence (IIF) technique, was extensively discussed. Cytoplasmic fluorescence pattern was found in patients with vasculitis. In contrast, perinuclear fluorescence pattern was found in vasculitis but also in many other inflammatory disorders. At the workshop, it was stated that IIF should be combined with an antigen-specific technique and capture and direct enzyme-linked immunosorbent assay techniques were discussed. In 1999/2003, an international consensus statement on testing and reporting on ANCA was published. For vasculitis, IIF was advocated as a screening assay, followed by an antigen-specific assay. In other non-vasculitic inflammatory diseases, only IIF on ethanol-fixed granulocytes was advocated. Recently, it was demonstrated that antigen-specific tests could be used as screening tests. Furthermore, it became clear that antigen-specific tests, in which antigens are directly coated to the solid phase, demonstrate highly variable sensitivities and specificities, whereas when capture or anchor technologies are used, more reproducible results are obtained. Based on these studies, we propose that the international consensus statement on ANCA should be revised.
Much has been learnt over the last 30 years to optimize the use of immunosuppressive and glucocorticoid therapies that has allowed the publication of treatment guidelines. However, major unmet needs remain in the treatment of ANCA‐associated vasculitis (AAV) and include refractory disease, only partial efficacy and toxicity of current drugs and the need for long‐term regimens. Newer therapies, including mycophenolate mofetil, leflunomide and rituximab, are providing a real opportunity for improved outcomes of AAV in the future. The development of therapy has been facilitated by international clinical research networks but delayed by the complexities of studying an uncommon, multi‐system disease.
Although repeatedly reported in the literature, the extracranial involvement by giant‐cell arteritis has been considered anecdotal until recent years. The emergence of new or improved imaging techniques along with a closer follow‐up of these patients and their increase in life expectancy are beginning to underline that the clinical impact of extracranial involvement by GCA may be more relevant than previously thought. This review focuses on the extent of vascular involvement in GCA as reported by pathology and imaging studies as well as the clinical consequences of imperfect vascular remodelling in various vascular territories.
Anti‐neutrophil cytoplasmic autoantibodies (ANCA) with a specificity for myeloperoxidase or proteinase 3 are closely associated with small vessel vasculitides (SVV). In vitro, ANCA activate primed neutrophils to release toxic substances that destroy endothelial cells, suggesting a pathogenic role for these autoantibodies in disease development. However, to study the complex interplay between ANCA, neutrophils, and the local environment in vivo, animal models are required. Here, we will review the animal models developed for ANCA‐SVV and discuss how these models have been applied to study ANCA‐SVV pathogenesis. In addition, some directions for future research pertaining to unresolved issues relevant for the pathogenesis and immunogenesis of ANCA‐SVV are proposed.
The inflammation of blood vessel walls that is associated with autoimmune disorders characterized by anti‐neutrophil cytoplasm antibodies (ANCA) represents dysregulation of normal physiological processes, whereby neutrophils recruited to the vessel wall by cytokine‐activated endothelium show destructive behaviours that initiate damage with endothelial apoptosis and denudation. Anti‐endothelial cell antibodies may also help to focus and escalate injury. Understanding the molecular mechanisms underlying the interplay between ANCA, aberrant neutrophil behaviour and vascular damage will allow development of more focused therapies.
The present study is the first complete presentation of the prognosis of all histological types of canine vascular tumours, using population‐based data. A retrospective, longitudinal, population‐based study of 420 dogs was carried out in 2001‐ 2002 using updated histologically verified diagnoses of vascular tumours and tumour‐like lesions recorded in Norwegian Canine Cancer Register (NCCR) 1990‐1998. Data on clinical signs and outcome of the vascular tumour, and causes of death, were obtained from dog owners and veterinary practitioners by mailed questionnaires. The a priori set inclusion criteria required that both questionnaire forms were completed, which was the case for 162 of the 420 dogs (39%). A benign tumour was diagnosed in 61%, whereas 15% had a tumour of intermediate malignancy and 24% had a malignant one. Survival time was estimated by the method of Kaplan‐Meier (product limit) survival plot, and the survival time for three histological diagnoses was compared by a log‐rank test. Hazard ratios were estimated by Cox proportional hazards model. A p‐value <0.05 was considered statistically significant. Median survival time of dogs with malignant vascular tumour was ≤one day, and the respective median survival times for intermediate malignant and benign types were 101 and ≥1095 days (P<0.0001). The median survival time of dogs with splenic vascular tumours was ≥1095 days, 110 days and ≤one day for benign, intermediate malignant and malignant tumour forms, respectively (P<0.0001). Since the histological diagnosis was made without any knowledge of the outcome, the present study confirms that the new histological criteria and methods employed have indeed a prognostic value. This is the first time it has been shown that a histopathological differentiation of malignant tumours into two well‐defined subgroups differentiates the survival time correspondingly: dogs with less grave tumour diagnoses live 100–110 days longer than dogs with the obvious malignant tumours, if surgery alone is the treatment regime.
The history of mankind over many millennia has been marred by many epidemics caused by viruses which infect the human respiratory system and alimentary tract. The current HIV-1/AIDS pandemic, however, is caused by one virus mutant, HIV-1M, which has evolved to infect humans through the genitals. The virus is able to use the innate system cells of the infected individual to inactivate the adaptive immune system, causing AIDS. The mechanisms used by HIV-1M to inhibit the immune system are presented. Understanding the viral mechanisms is leading to novel antiviral treatments and an approach to an HIV-1 vaccine.
* Disclaimer: the article reflects the author’s but not the institution’s opinion (see also http://www.rki.de). This article is in part an evaluation and assessment of results which will be published elsewhere in detail. Contributions of unpublished raw data included here were from the Master Theses of Thomas Willemsen (epitopes on BDV Nand P-protein), Dennis Schmidt (BDV N-protein characterisation from plasma), and Steffen Zander (BDV P-protein). Corresponding author: E-mail (office): bodel/rki.de E-mail (Internet): Liv.Bode/web.de
