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M. hominis is commonly found as part of the normal flora in the female genital tract, but several studies have shown that it may be involved in a variety of urogenital infections. The basis for clinical manifestations in some patients has varyingly been attributed to host and M. hominis factors. The host factors involved in the infection process are largely unknown. M. hominis have no cell wall and outer membranes, and at present it seems plausible that M. hominis possesses genetic systems allowing the bacteria in vivo to alter its antigenic structure on the membrane surface and consequently circumvent the host immune system. The studies of M. hominis have shown that the antigenic variation is pronounced between surface exposed membrane proteins from different isolates. The genetic background for this variation has been investigated for three surface exposed membrane proteins: P120, Lmp, and Vaa. P120 and P120' are similar proteins in M. hominis without any homology to other known proteins. A hypervariable region in the otherwise conserved P120 protein seems to be very antigenic in patients with immunologically verified M. hominis infection. The remaining part of P120 as well as the entire P120' protein do not seem to elicit significant antibody formation. Two genes in M. hominis, lmp1 and lmp3, contain numerous highly similar 0.5 kb tandem repeats at their 3'-end. The proteins, Lmp1 and Lmp3, are synthesized from the lmp1 and lmp3 genes, respectively. Lmp1 shows size variation among M. hominis isolates. M. hominis isolates investigated in detail show that the size variation of Lmp1 corresponds to the variation in number of 0.5 kb repeats contained within the lmp1 gene. Lmp3 appears to have a lesser tendency to size variation. M. hominis isolates were found with deletions involving the lmp1 stop codon leading to translation of the downstream gene lmp2 and expression of a chimeric Lmp1-Lmp2 protein. The number of repeated elements in the lmp1 gene of a M. hominis isolate correlates with the extent of anti-Lmp antibody induced agglutination between the bacteria. Vaa is a protein involved in cell adherence. vaa is a single copy gene containing tandem repeated elements like the lmp gene family. The number of repeats in the Vaa protein differs between M. hominis isolates leading to size variation. It has been suggested that the number of repeated elements is of importance in the bacteria-host adhesion process. Beside the size variation Vaa demonstrates phase variation due to frequent frame shift mutation in a specific region near the 5'-end of the structural gene. Based on the investigations of M. hominis and other mycoplasmas several genetic mechanisms seem to be responsible for the antigenic variation of surface exposed membrane proteins in mycoplasmas: 1) variation in protein size due to insertions or deletion of repeated elements in the structural gene, 2) presence of multi-gene families, and 3) phase variation due to mutations in the promot

Unlabelled: 14.1

Introduction: This thesis is based on a number of monitoring and research programmes initiated at the Danish Veterinary Laboratory with the aim to determine the occurrence, selection and spread of resistance to antimicrobial agents used for growth promotion. The thesis gives a brief overview of the use, consumption, function and benefit of antimicrobial growth promoters and a more thorough description of the potential resistance problems arising by the use of these agents. 14.2 THE USE OF ANTIMICROBIAL AGENTS IN A HISTORICAL PERSPECTIVE: Soon after the introduction of antimicrobial agents for therapy of bacterial infections in humans and animals, the growth promoting effect of antimicrobial agents was observed, and since the beginning of the 1950'ties antimicrobial agents have been included in feed for food animals as a way to improve growth and reduce production costs. 14.3 CONSUMPTION OF ANTIMICROBIAL GROWTH PROMOTERS: Exact figures on the consumption of antimicrobial agents for clinical and growth promoting purposes are very difficult to get, and estimates are only available for a few countries. In Denmark, the total annual consumption of antimicrobial agents for growth promotion increased from 67 tonnes to 116 tonnes from 1989 to 1995. After the ban on avoparcin in 1995 the total consumption of growth promoters decreased to 94 tonnes. An increase up to 107 tonnes took place during 1996 and 1997, but during 1998, the consumption decreased to approximately 49 tonnes. The data that are available for different countries show that the use of antimicrobial agents for growth promotion normally equals or exceeds the usage of antimicrobial agents for therapy for food animals. Based on the information available, it can be estimated that the financial sale of antimicrobial agents for animals amounts to approximately 25% to 35% of the world-wide sale, of which the use of antimicrobial agents as feed additives is at least 50%. 14.4 MODE OF ACTION OF ANTIMICROBIAL GROWTH PROMOTERS: The mode of action of antimicrobial growth promoters is not fully understood. However, the main effects are believed to be a reduction of the growth of bacteria in the intestinal tract and thereby less microbial degradation of useful nutritients, and the prevention of infections with pathogenic bacteria. 14.5 BENEFIT FROM THE USE OF ANTIMICROBIAL GROWTH PROMOTERS: Numerous studies on the economic benefit of the use of antimicrobial growth promoters have been performed. The growth response is normally larger in young animals than in older animals. Large variations in the estimates have been observed, but in general a improvement in growth rate and feed utilisation has been observed. 14.6 SUSCEPTIBILITY AND RESISTANCE TO ANTIMICROBIAL GROWTH PROMOTERS: The definition of a bacterium as susceptible or resistant to an antimicrobial agent ultimately depends on clinical outcome. Since the exact mode of action of antimicrobial

Periodontal diseases affect millions of people world wide. Prevention and treatment of these diseases require considerable attention from the individual as well as society and cause great expenses. Understanding disease etiology and mechanisms of pathogenesis is a prerequisite for optimal treatment strategies. The highly variable speed of periodontal destruction and in some sites persistence for years of deep pockets without further periodontal destruction points to the significance of individual bacterial species in the complex subgingival microflora for pathogenesis. Destruction of periodontal tissue occurs when the load of bacterial virulence factors overcomes the local immune defense. One way of doing this is by bacterially-induced degradation of IgA which is considered to mediate its protective functions in an anti-inflammatory way and to down-regulate inflammation through inhibition of IgG- and IgM-mediated activities. A competent IgA system may be of particular significance in chronic inflammatory diseases, as periodontal diseases, where the inflammatory reaction in itself probably is the main cause of destruction. In these cases, degradation of IgA may serve the purpose of immune evasion for the bacteria and at the same time may induce a relatively increased activity in the inflammation-stimulating part of the immune system which may aggravate periodontal destruction. Both gram-positive rods, streptococci, and Veillonella species from the subgingival microflora induce an altered immunoelectrophoretic mobility of IgA1 indicative of removal of terminally positioned sialic acid. Quantitative determination of residual carbohydrate content of IgA1 after incubation with bacterial cells of Gram-positive rods has confirmed that they remove sialic acid, and in addition to that, only minor amounts of carbohydrates. Apart from serving a nutritional purpose, desialylation of IgA may also serve a purpose of immune evasion. Glycosylation and, in particular sialic acid protects glycoproteins, including immunoglobulins, against proteolytic enzymes and deglycosylation of antibodies increase their sensitivity to proteolytic degradation and inhibit the Fc-mediated effector functions that mediate antigen disposal. Extensive proteolytic degradation of IgA1 is induced by a number of bacterial species often associated with periodontal diseases, including P. gingivalis, Pr. intermedia, and Pr. nigrescens. These species produce enzymes of broad proteolytic activity, that also may degrade immunoglobulins of other isotopes, complement factors, iron-containing plasma proteins etc. Extensive hydrolysis of immunoglobulins induced by these bacteria serve a nutritional purpose and is essential for growth of other bacteria in mixed cultures. It also has an obvious detrimental effect on the defence potential of the specific humoral immune system. These bacteria seem to be essential for the transmissibility of experimental infections in animals with mixtures of oral

Numerous studies on DNA amplification and diagnosis of C. trachomatis infections have been performed since the first PCR for detection of C. trachomatis in clinical samples was described in 1990, but optimal sample preparation procedures are still lacking. The major problem in evaluating the diagnostic performance of the DNA amplification methods is that there is no clinical or microbiological reference standard for C. trachomatis infection. The evaluated diagnostic performance will therefore always be a reflection of of the chosen comparator(s). Despite this, it seems that the DNA amplification methods are more sensitive than the cell culture techniques and the techniques based on immunology. Compared with the tests based on immunology the specificity is also improved, which makes the DNA amplification methods useful for sample types contaminated with organisms cross-reacting in the immunologically based methods, i.e. pharyngeal and rectal swab samples. However, the specificity and thereby the predictive value of a positive test is not optimal. Since the predictive value of a positive test is highly influenced by the prevalence in the tested population, evaluation of applied tests is constantly needed, especially since the prevalence may be expected to decrease with intensified test activity and due to changes in safe sex practices after the advent of AIDS. The improved sensitivity of the DNA amplification methods allows the use of sample material in which the content of Chlamydia organisms is lower than in conventional swab samples, i.e. urine, semen, and vaginal secretions can be used as sample material. these samples can be obtained by the individuals themselves, and since transport conditions seem less critical for the test performance, samples can also be taken in the privacy of the home and subsequently mailed by the individual directly to the diagnostic laboratory. Such strategy for testing has led to improved partner tracing and universal screening, compared with traditional swab examinations at physicians' offices. Tests with an optimal diagnostic performance have not yet been reached, and several sample categories have not been studied sufficiently. The societal implications of the use of self-collected samples and universal screening have not been studied in full, but a milestone for new strategies in detection and preventing urogenital C. trachomatis epidemics has been reached with the availability of DNA amplification techniques.

Epidermal growth factor (EGF) belongs to a family of growth factor ligands and receptors. At present, five ligands have been recognized which as EGF exert their effects via binding to the same EGF receptor. The family has three other receptors erbB2, erbB3, and erbB4, which have their own ligands (the heregulins). The system is ubiquitously distributed in mammals, and has important roles in normal development, and in regenerative and neoplastic growth. Mouse and human EGF were discovered in 1962 and 1975 by Stanley Cohen and Harry Gregory, respectively, due to EGFs potent systemic effects. EGF accelerated eyelid opening in newborn mice and inhibited gastric acid secretion in humans. Already in the late thirties, a factor in human urine was recognized which prevented or accelerated healing of experimental damage in the gastrointestinal tract. This factor appeared to be EGF. Around 1980, an effect of commercial interest was described-EGF caused shedding of the fleece in sheep. In line with the original observations, several studies have examined effects of EGF on developmental processes. Amongst other effects, EGF accelerates lung and intestinal maturation before birth and in newborn mammals. Due to the possible use of EGF in the wool industry, it was mandatory to know more about EGF. Amongst other effects in mature sheep and other animals are haemodynamic changes, changes in electrolyte homeostasis, and endocrinological changes. In relation to experimental damage, the therapeutic potential of systemic EGF has been demonstrated in all parts of the gastrointestinal tract, in the kidneys, in the liver and in the trachea. EGF has even been tried in humans in gastric ulcer healing and in necrotising enterocolitis. Studies on prolonged treatment with EGF have first recently appeared. We described effects of 4-5 weeks of treatment in Goettingen minipigs and in rats, and two other groups described effects in monkeys and in rats. In summary, species differences were observed. The species of higher order were most sensitive to treatment with EGF. EGF did not consistently change the total body weight despite EGF consistently reduced circulating levels of insulin-like growth factor I (IGF-I) in Goettingen minipigs as well as in rats. Low circulating levels of IGF-I are usually associated with retarded growth. This review mostly focuses on the organs which appeared to be most sensitive to EGF, the urinary and gastrointestinal tracts including the liver and the pancreas. The histopathological changes consisted mainly of epithelial proliferations in the gastrointestinal, urinary and respiratory tracts. These findings match the knowledge obtained from animals overexpressing the EGF agonist, transforming growth factor alpha (TGF alpha), and the mice with a knock out of the gene encoding for the EGF receptor. EGF receptor hyperstimulation (TGF alpha overexpression) in the context of the whole animal leads to epithelial proliferations whereas hypostimulation (

This thesis describes new and original experimental results on Cu-dependent amine oxidases (CAOs), which show that these enzymes can be conveniently and specifically detected in situ using a peroxidase-coupled activity staining method with 4-Cl-1-naphtole as hydrogen donor substrate. Even more sensitive in situ detection can be achieved using a chemiluminescence-based coupled peroxidase assay which was applied to show that human placenta CAO activity is confined to maternal vessels. A general purification scheme for CAOs is described, and applied to purification of different CAOs. Peptide maps and immunological crossreactivity studies with monoclonal antibodies raised against the purified enzymes showed that they were closely related. Amino acid sequence data for the bovine serum CAO showed that they form a separate group (E.C. 1.4.3.6) with no homology to other enzymes. A cDNA sequence was obtained on the basis of the amino acid sequence data, and this was found to encode a bovine lung CAO, related to bovine serum CAO. The genes for bovine lung and bovine serum CAO are characterized, and Southern blotting analysis of bovine chromosomal DNA shows the existence of a least one more bovine CAO. The purification of human neutrophil CAO is attempted, but it is described how lactoferrin, a protein with many properties in common with CAOs, and with a low degree of sequence identity can account for many observations on human neutrophil CAO. The products of bovine serum CAO oxidation of polyamines are characterised, and 3-aminopropanal is found to be the principal aminoaldehyde produced. Finally, a polyamine-stimulated binding of human placenta CAO to single-stranded DNA is described, and it is reported that the DNA-bound CAO is enzymically active and that the oxidation of DNA-bound polyamines leads to degradation of DNA. In addition to the experimental results, the properties of polyamines and Cu-dependent amine oxidases are reviewed. The polyamines spermidine and spermine interact specifically with nucleic acids and several other molecules. They are synthesised from putrescine, which is a key regulatory molecule formed from ornithine by ornithine decarboxylase, a highly inducible and regulated enzyme. The polyamines can be converted to putrescine by CAOs or spermidine/spermine acetyltransferase and polyamine oxidase. Putrescine is degraded by CAOs, which are also involved in degradation of histamine, a mediator of inflammatory processes. CAOs catalyse the general reaction: R1CH2NHR2 + O2 + H2O-->R1CHO + R2NH2 + H2O2 and in addition to the catabolism of putrescine and histamine CAOs are involved in regulation of growth and apoptosis by to the generation of aminoaldehydes and hydrogen peroxide which have growth inhibitory properties. Several homologous CAOs have been purified and characterized and they form a family with two subgroups. They are homodimers with a relative molecular weight of 180,000 and contain Cu2+ and a modified tyrosine, topaquino