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Effect of surgical intervention on the activation status of circulating monocytes and T-cells
(2009)
Major surgery causes alterations in immune function which results in immune suppression in post surgical patients. Deactivation of monocytes in these patients is characterised by the reduced ability of these cells to produce pro-inflammatory cytokines on stimulation with LPS in vitro and by markedly reduced HLA-DR expression. Immune suppression in patients with systemic inflammation has also been associated with a high level of apoptosis in both the circulating T and B cell populations. In addition post surgical T cells have a reduced capacity to proliferate ex vivo in response to co-ligation of the T cell receptor and CD-28. Considering these impairments of immune system, this study aimed to define the extent of immune modulation in both innate and adaptive system in a cohort of surgical patients. Measurment of the level of HLA-DR expression of monocytes in these patients showed a considerable change in monocyte phenotype in the immediate post operative period. In line with previous work, all patients showed a considerable reduction in monocytic surface HLA-DR expression which persisted for many hours and those who had post surgical septic complications showed the most severe reduction. Importantly, patients with minor surgical intervention also exhibited decreased HLA-DR expression. Gene expression analysis of monocyte in these patients showed the up-regulated transcripts of genes involved in extravasation and realignment of the cytoskeleton. Analysis of periperal T cell demonstrated a significant reduction in their number in the circulation and a sharp raise in the number of apoptotic T –cells in the immediate post surgical period. Microarray analysis of T cells from patients who developed sepsis and patients with an uneventful recovery within the post-operative period (3 days) showed a substantial reduction in the transcriptional activity of many genes in both groups. However, this down regulation of T cell transcriptional activity appears to be a rather broad and non specific effect since it is not restricted to particular functional pathways. Real time PCR analysis of both the CD4+ and CD8+ populations using selected down-regulated genes showed that the change in transcriptional profile is equally evident both in CD4+ and CD8+ T-cells. The cause of this transient immune depression following surgery remains to be established and it may represent an important enabling factor which contributes to the development of post surgical infections and inflammatory complications.
SUMMARY To date, Staphylococcus aureus is the most common cause of nosocomial infections and the species is becoming increasingly resistant to antibiotics. Beyond this, S. aureus colonises the nasal mucosa of circa 35% of the healthy population, so-called carriers. Importantly, S. aureus nasal carriage is a major risk factor for the development of S. aureus infections, which are commonly caused by the colonising strain. This underlines the importance of host factors for the outcome of S. aureus-host interactions. Despite the clinical importance of nasal carriage, little is known about humoral immune responses triggered by colonisation. Therefore, this thesis was focussed on the anti-staphylococcal antibody responses of S. aureus carriers and noncarriers. Staphylococcal superantigens (SAgs) served as indicator antigens for our studies. SAgs are virulence factors with extraordinary variability in the species S aureus and act as extremely potent T cell mitogens. To date, 19 different SAg gene loci are known in the species S. aureus, but molecular-epidemiological studies on the distribution of these genes are limited. Therefore, we established five multiplex PCRs for the detection of all known SAgs. With this robust and high-throughput technique we analysed the SAg gene patterns of more than 300 isolates, including 107 nasal isolates of S. aureus carriers and 88 blood culture isolates of hospital patients from Western Pomerania. The SAg gene patterns were highly heterogeneous, which can be explained by their localisation on mobile genetic elements (MGE), such as genomic islands, pathogenicity islands, phages and plasmids. Most isolates (~80%) harboured SAg genes, on average five to six, and SAgs of the enterotoxin gene cluster (egc) were by far the most prevalent. Additionally, we observed a strict correlation between the presence of SAg genes and the T cell mitogenic potency of clinical isolates. SAg-encoding MGEs can be distributed by two distinct mechanisms: horizontal transfer by bacteriophages and vertical transmission to daughter cells. To investigate the distribution of SAg genes within the S. aureus population, we determined the clonal relationship of our isolates by spa genotyping. Interestingly, SAg-gene encoding MGEs were not randomly distributed, but rather closely linked to clonal lineages. Each clonal lineage was characterised by defined combinations of SAg genes. These data suggest that the simultaneous assessment of virulence gene profiles and the genetic background strongly enhances the discriminatory power of genetic investigations into the mechanisms of S. aureus virulence. Indeed, the comparison of virulence genes within each clonal complex indicated a role in invasiveness for some MGEs, e.g. the exfoliative toxin D-encoding pathogenicity island, while rendering it unlikely for SAgs. It is known that neutralising serum antibodies against the SAgs SEA, SEB, SEC, SED and TSST-1 are frequently present in healthy individuals. However, the neutralising antibody profiles against more recently described SAgs or complex SAg cocktails as secreted by clinical isolates had not been determined so far. Therefore, we screened more than 100 sera for their SAg neutralising capacity with a neutralisation assay. We observed a marked heterogeneity and surprisingly large “gaps” in the neutralising capacity. Interestingly, the egc SAgs were inhibited only rarely (5-10%), whereas between 32 and 86% of the tested sera neutralised “classical” SAgs. This “egc gap” in the SAg-neutralising antibody profiles of healthy individuals was unexpected, since egc SAgs are by far the most prevalent SAgs. We could demonstrate that the “egc gap” is probably not due to different T cell activating properties of egc SAgs compared to classical SAgs, but rather to a differential regulation of SAg gene expression. S. aureus carriers have an increased risk of developing an S. aureus bacteraemia, which is in most cases caused by the colonising strain. Intriguingly, a large prospective clinical trial revealed a considerably higher mortality in noncarriers with invasive S. aureus strains compared to carriers with invasive disease. To explain these paradoxical findings, we hypothesised that in carriers partial immunity against the colonising strain may contribute to their improved outcome. We used SAgs as strain-specific indicator antigens. Importantly, sera from persistent carriers neutralised SAgs of their colonising strain with significantly higher efficiency than sera from noncarriers. This antibody response was strain-specific, since the antibody response of carriers against other SAgs did not differ from that of noncarriers. Thus, colonisation with S. aureus confers a strong and strain-specific antibody response against staphylococcal SAgs. We suggest that in carriers neutralising antibodies directed against SAgs and other staphylococcal virulence factors confer partial protection during systemic infections. This could explain the better prognosis of carriers with S. aureus bacteraemia compared to noncarriers. Moreover, our data imply that the key to understanding the pathogenesis of S. aureus disease may lie in the identification of host factors rather than bacterial factors. Such host factors could be the immune status and gene polymorphisms that contribute to colonisation, susceptibility to infection and outcome of infection. Finally, while the treatment of S. aureus bacteraemia with pooled immunoglobulins was performed in the past without significant success, our findings on strain-specific antibody profiles suggest that therapies with customised cocktails of monoclonal antibodies could have a higher efficacy.