Mechanisms of Bacterial Colonization of Implants and Host Response.

The review focuses on the current knowledge and the most pertinent hypotheses regarding the local host immune response as the key factor for the pathogenesis of implant-associated infections. Although bacterial biofilms have long been recognized as causative agents, the link between the infection and the devastating inflammatory response, particularly the localized tissue destruction and bone degradation is less well understood. Understanding these consequences of infection, however, is of utmost importance, because suppressing inflammation and preventing bone destruction could be a novel, alternative therapeutic option in cases when eradicating the infections fails.

Human polymorphonuclear neutrophils express RANK and are activated by its ligand, RANKL.

The receptor activator of NF-κB (RANK) is especially well studied in the context of bone remodeling, and RANK and its ligand, RANKL, are key molecules in the induction of bone resorbing osteoclasts. We now report that polymorphonuclear neutrophils (PMNs) contain preformed RANK, stored in secretory vesicles and in specific granules. Upon stimulation of PMNs in vitro, RANK was translocated to the cell membrane. In patients with persistent bacterial infections, RANK surface expression was enhanced compared with that of healthy individuals. The functional activity of RANK was assessed by determining migration of PMNs toward RANKL. A time- and dose-dependent migration was seen, leading to the conclusion that RANK on PMNs is functional. We presume that regulated RANK expression contributes to the fine tuning of PMN migration, for example, on and through inflamed endothelium that is known to express RANKL.

Inhibition of osteoclast generation: a novel function of the bone morphogenetic protein 7/osteogenic protein 1.

Monocytes have the potential to differentiate to either macrophages, dendritic cells, or to osteoclasts. The microenvironment, particularly cytokines, directs the monocyte differentiation. Receptors of NFκB (RANK) ligand, tumor necrosis factor (TNF) α, or interleukin- (IL-) 8 have be identified as inducers of osteoclastogenesis, whereas others, such as IL-10 or transforming growth factor (TGF)ß inhibit osteoclast generation or induce differentiation towards a dendritic cell type. We now describe that bone morphogenetic protein (BMP) 7/osteogenic protein- (OP-) 1 inhibited the differentiation of human CD14+ monocytes to osteoclasts. In the presence of BMP7/OP-1 the transcription factors c-Fos and NFATc1, though upregulated and translocated to the nucleus in response to either RANKL or IL-8, did not persist. In parallel, MafB, a transcription factor expressed by monocytes and required for differentiation to macrophages but inhibiting osteoclast generation, was preserved. Because both persistence of NFATc1 and downregulation of MafB are crucial for osteoclastogenesis, we conclude that BMP7/OP-1 inhibits the generation of osteoclasts by interfering with signalling pathways.

Palladium(II) ortho-cyano-aminothiophenolate (ocap) complexes.

A series of Pd(II) complexes containing ortho-cyano-aminothiophenolate (ocap) ligands have been prepared and their molecular structures elucidated. Hg(II) ocap complexes, [Hg{SC6H3XN(CN)}]n (X = H, Me) (1), react with Na2S to afford HgS and Na2[ocap] which reacts in situ with K2[PdCl4] to afford palladium ocap complexes [Pd{SC6H3XN(CN)}]n (2). A second route to these coordination polymers has also been developed from reactions of 2-aminobenzothiazole (abt) complexes, trans-PdCl2(abt)2 (3), with NaOH. We have not been able to crystallographically characterise coordination polymers 2, but addition of PPh3, a range of phosphines and cyclic diamines affords mono and binuclear complexes in which the ocap ligand adopts different coordination geometries. With PPh3, binuclear [Pd(µ-κ2,κ1-ocap)(PPh3)]2 (4) results, in which the ocap bridges the Pd2 centre acting as an S,N-chelate to one metal centre and binding the second via coordination of the cyanide nitrogen. In contrast, with diphosphines, Ph2P(CH2)nPPh2 (n = 1-4), mononuclear species predominate as shown in the molecular structures of Pd(κ2-ocap){κ2-Ph2P(CH2)nPPh2} (5-7; n = 1-3). With 2,2'-bipy and 1,10-phen we propose that related monomeric chelates Pd(κ2-ocap)(κ2-bipy) (9) and Pd(κ2-ocap)(κ2-phen) (10) result but we have been unable to substantiate this crystallographically. Addition of HgCl2(phen) to 9a (generated in situ) affords heterobimetallic Pd(κ2-phen)(µ-κ2,κ1-ocap)HgCl2(κ2-phen) (11), in which Hg(II) is coordinated through the ring sulfur.

Expression of granzyme B in peripheral blood polymorphonuclear neutrophils (PMN), myeloid cell lines and in PMN derived from haemotopoietic stem cells in vitro.

Granzyme B and perforin are the major protagonists of cytotoxicity mediated by natural killer (NK) cells or cytotoxic T cells. More recent we described the presence of granzyme B and perforin in polymorphonuclear neutrophils (PMN), a finding in discrepancy with the credo that granzyme B and perforin expression is restricted to cytotoxic T cells and NK cells. In extension of our previous study, we now provide evidence that granzyme B is not only present in mature PMN, but also in the myeloid cell lines HL-60 and U937, in CD34+ stem cells, and in PMN derived from CD34+ cells in vitro. In agreement with the "targeting by time" hypothesis we found the bulk of granzyme B in association with primary granules, in addition to a minor membrane expression. Granzyme B, on one hand might, enhance the cytotoxic potential of PMN, on the other, it may provide PMN with additional means to degrade extracellular matrices.

Lipopolysaccharides (LPS) induce the differentiation of human monocytes to osteoclasts in a tumour necrosis factor (TNF) alpha-dependent manner: a link between infection and pathological bone resorption.

The degradation of bone is a serious consequence of persistent bacterial infection, including periodontitis, infection-associated non-unions or osteomyelitis. To test the hypothesis that infection and inflammatory conditions promote the differentiation of monocytes to bone-resorbing osteoclasts, highly purified monocytes, or alternatively, cells of the promyeloid cell line U937, differentiated to monocyte-like cells, were cultivated in the presence of lipopolysaccharides (LPS) for up to 30 days. After 2-4 days, a massive aggregation of the cells was observed, after 15-20 days multinuclear cells with the morphological characteristics of osteoclasts became apparent. These cells expressed the osteoclast-typical proteins tartrate-resistant acid phosphate (TRAcP) and cathepsin K. Moreover, these cells formed resorption pits on calcium phosphate coated cover slips or ivory slices. To test whether the differentiation of the monocytes to osteoclast-like cells was mediated by tumour necrosis factor alpha (TNFalpha) secreted by the cells in culture, an antibody directed against TNFalpha was added together with LPS. Differentiation to osteoclast-like cells was inhibited, suggesting a paracrine effect of locally produced TNFalpha. In conclusion, we propose that local bacterial infections could create a microenvironment that promotes the generation of bone resorbing cells, which, in turn, could contribute to the infection-associated osteolysis.

T lymphocytes in acute bacterial infection: increased prevalence of CD11b(+) cells in the peripheral blood and recruitment to the infected site.

T-cell activation, particularly of CD8(+) cells, is invariably associated with viral infections. We now provide evidence for the activation of T cells in patients with localized bacterial soft tissue infections. During acute disease we detected in the peripheral blood of these patients, small though conspicuous populations of CD4(+) CD28(+) CD11b(+) and CD8(+) CD28(+) CD11b(+) cells, indicative of an expansion of effector T cells. Moreover, we identified CD4(+) and CD8(+) cells at the infected site, in addition to highly activated polymorphonuclear neutrophils (PMN). In keeping with their role as first-line defence, PMN were preponderant, but T cells amounted to 20% of the infiltrated cells. The majority of the infiltrated T cells expressed CXCR6, a homing receptor for non-lymphoid tissue. The infiltrated T cells produced interferon-gamma (IFN-gamma), while the peripheral blood cells obtained at the same time did not. In conclusion, in response to localized bacterial infections, T cells are activated and recruited to the infected site. We propose that these T cells, e.g. by producing IFN-gamma, enhance the efficiency of the infiltrated phagocytic cells, particularly of the PMN, thereby supporting the local host defence.

Receptors for complement C3 on T-lymphocytes: relics of evolution or functional molecules?

Despite the fact that receptors for complement on T-cells have been described many years ago the function remains unclear as is the role of complement in the T-cell response. In this review we will evaluate how the accumulated wisdom concur with the current concepts of the adaptive T-cell response.

The complement receptor 1, CR1 (CD35), mediates inhibitory signals in human T-lymphocytes.

The modulation the specific, adaptive immune response by complement, particularly of by complement C3, is mainly attributed to its interaction with complement receptors on B-lymphocytes. The function of complement receptors on T-lymphocytes, in contrast, is less well understood, although expression of the complement receptor (CR)1 and CR3 on T-cells has been described years ago. In the present study we investigated the effect of antibodies to CR1 on T-cell lines and peripheral T-cells of healthy donors, respectively. Antibodies to CR1 profoundly inhibited the proliferation of the T-cells; of note is, that exogenously added interleukin 2, though enhancing proliferation, did not overcome the inhibitory effect mediated by anti-CR1. While anti-CR1 had no effect on the activation of the immediate early genes c-jun or c-fos nor on the early increase of gamma interferon- or interleukin 2-specific RNA, the protein synthesis of those cytokines was inhibited. Moreover, synthesis of the proliferating cell nuclear antigen (PCNA) was reduced as was the expression of cyclins, particularly of cyclin A and cyclin D3. Taken together, the data indicate that triggering CR1 inhibits proliferation of T-lymphocytes by a mechanism operating downstream of the initial signalling events.

T lymphocytes in implant-associated posttraumatic osteomyelitis: Identification of cytotoxic T effector cells at the site of infection.

In implant-associated posttraumatic osteomyelitis, a massive infiltration of leukocytes into the infected site is seen. As described previously, the most infiltrated cells were highly activated polymorphonuclear neutrophils. In addition, a considerable T-cell infiltrate was noted. Whereas our previous work was mainly concerned with the phenotypical and functional characterization of the polymorphonuclear neutrophils, we now analyzed T lymphocytes of 32 patients with implant-associated posttraumatic osteomyelitis. We found evidence for an expansion of CD8 T cells in the peripheral blood of the patients and for an infiltration of these cells into the infected site. Further analysis of the surface-receptor pattern by three-color cytofluorometry revealed that the majority of these cells belonged to the cytotoxic-effector phenotype. Of note is that cytotoxic T cells are generally associated with virus infection. Thus, the detection of those cells in patients with bacterial infection was rather unexpected and points to a novel, not yet appreciated, role of CD8 T cells also in the defense of bacterial infections.

Expression of elastase on polymorphonuclear neutrophils in vitro and in vivo: identification of CD11b as ligand for the surface-bound elastase.

Elastase is a major serine protease of polymorphonuclear neutrophils (PMN). On activation of PMN, the preformed protein is mobilized from intracellular stores and, depending on the activating conditions, is either released into the supernatant or is bound to the cell surface. By a variety of methods, including uptake and crosslink studies, as well as confocal laser scan microscopy, we now provide evidence that elastase binds to the beta(2)-integrin CD11b and induces a conformational alteration of CD11b, apparent as expression of a neodeterminant. Similarly to the in vitro data, elastase surface expression and conformational alterations of CD11b were seen on PMN of patients with Staphylococcus aureus-induced localized infection, particularly on PMNs recovered from the infected site. The presence of elastase at the site of inflammation is in keeping with its presumed role in leukocyte trafficking and host defense. On the other hand, because of its potential for degrading extracellular matrix proteins, elastase could participate in localized tissue damage as it occurs in severe S. aureus infection.

Post-traumatic osteomyelitis: analysis of inflammatory cells recruited into the site of infection.

Device-associated infections after implants or endoprostheses inflict local inflammation and ultimately osteolysis, a clinical entity referred to as posttraumatic osteomyelitis. The underlying molecular mechanisms are not yet known; formation of bacterial biofilms on the implant is presumed, conferring resistance to antibiotics and to host defense mechanisms as well. To gain insight into the pathogenesis of post-traumatic osteomyelitis, the infected site was analyzed for the presence of immunocompetent cells. In 18 patients, the infected site was rinsed intraoperatively. This so-called lavage contained 1-2 x 107 leukocytes, predominantly highly activated polymorphonuclear neutrophils (PMNs), as characterized by low expression of CD62L (selectin), and high expression of the adhesion protein CD18, of the high-affinity immunoglobulin (IgG) receptor CD64, and of the LPS-receptor CD14. CD16, the low-affinity IgG receptor, was affected in some patients only. Because the majority of infections were caused by staphylococci species, the effect of bacteria-derived lipoteichoic acid on PMN of healthy donors was tested in vitro. A similar activation pattern was found: rapid down-regulation of CD62L, a slower loss of CD16, and upregulation of CD18, CD64, and CD14. Lipoteichoic acid signaling required p38 mitogen-activated protein kinase and resulted in induction of CD14-specific mRNA and de novo protein synthesis. We conclude that PMNs infiltrate the infected site, but despite local activation they are unable to clear the bacteria, presumably because of biofilm formation. Our data are consistent with the hypothesis that during the ineffective "frustrated" attempt to phagocytose, PMNs release cytotoxic and proteolytic entities that in turn contribute to the progression of tissue injury and ultimately to osteolysis.

Polymorphonuclear neutrophils in posttraumatic osteomyelitis: cells recovered from the inflamed site lack chemotactic activity but generate superoxides.

The pathogenesis of posttraumatic osteomyelitis, one of the major complications after orthopedic surgery, is not yet understood. Formation of bacterial biofilms on the implant is presumed, conferring resistance to antibiotic therapy and probably also to the host defense mechanisms. In that context, the polymorphonuclear neutrophils (PMN) having infiltrated the infected site were recovered and characterized phenotypically and functionally. Loss of CD62L and upregulation of CD14 were seen, as was expression of CD83. Expression of the latter is dependent on de novo protein synthesis and thus is indicative of an extended life span and a transdifferentiation of the PMN at the infected site. The infiltrated PMN had lost their chemotactic activity, whereas the capacity to produce superoxides was preserved and in some patients even enhanced. In vitro experiments done in parallel showed that long-term culture with interferon-gamma resulted in similar alterations of PMN: loss of chemotactic activity, whereas other functions of PMN, such generation of superoxides and phagocytosis of opsonized bacteria, were preserved or even enhanced. The loss of the migratory capacity of PMN having already emigrated from the blood vessel to the infected site is not expected to affect the host defense negatively. Assuming, however, that bacteria are organized as a biofilm and that infiltration into this biofilm is required for phagocytosis of the bacteria, our data could to some extent explain why despite being activated, the PMN are not able to control the infection. By releasing their cytotoxic, proteolytic, and collagenolytic potential, PMN might instead contribute to tissue destruction and eventually to osteolysis.

Expression patterns of the lipopolysaccharide receptor CD14, and the FCgamma receptors CD16 and CD64 on polymorphonuclear neutrophils: data from patients with severe bacterial infections and lipopolysaccharide-exposed cells.

In polymorphonuclear neutrophils (PMN) CD14, one of the receptors for lipopolysaccharides (LPS) is stored intracellularly as a preformed protein, with only few receptors expressed on the surface. We now report that in patients with severe bacterial infections, CD14 expression is profoundly upregulated, as is CD64 (FcgammaRI), the high-affinity receptor for IgG, whereas CD16 (FcgammaRIII) was partly lost from the surface. To further analyze regulation of these receptors, PMN of healthy donors were exposed to low doses of LPS. By brief exposure (10-120 min) to LPS, CD14 was transferred to the surface in a cytochalasin B-sensitive manner, as were CD16 and CD64. Prolonged culture (up to 48 h) resulted in a further upregulation of CD14, sustained expression of CD64, and profound decline of CD16, yielding a similar pattern of receptor expression as seen in the patients. Subsequent studies revealed that LPS induced de novo synthesis of CD14: the increase of surface expression could be inhibited by cycloheximide and by interfering with a known LPS-induced signaling event, the translocation of NFkappaB. Moreover, an up to 10-fold increase of specific mRNA was seen, as was incorporation into CD14 of 35S-methionine. The de novo synthesis prolonged expression of CD14, whereas the CD16 expression declined, generating a PMN phenotype characteristic for severe infection and indicative of escape from apoptosis of a PMN subpopulation.

Attitudes toward lesbians, gay men, bisexual women, and bisexual men in Germany.

Attitudes toward lesbians, gay men, bisexual women, and bisexual men were assessed in a national representative sample of 2,006 self-identified heterosexual women and men living in Germany. Replicating previous findings, younger people held more favorable attitudes than older people; women held more favorable attitudes than men; and men held more favorable attitudes toward female than male homosexuality, whereas women did not differentiate. However, women held more favorable attitudes toward homosexuals than toward bisexuals, whereas men did not differentiate. Knowing a homosexual person was an important predictor of attitudes, as was political party preference. Both same-sex and opposite-sex sexual attraction were substantially related with attitudes. Our findings support the notion that attitudes toward lesbians, gay men, bisexual women, and bisexual men are related but distinct constructs.

Biofilm growth on implants: bacteria prefer plasma coats.

PURPOSE: Bacterial biofilm formation on prostheses or devices used for osteosynthesis is increasingly recognized as cause of persistent infections, an entity known as implant-associated posttraumatic osteomyelitis. Biofilm formation is a very complex, multistep process with adhesion as the first and decisive step. The most prevalent pathogens found are staphylococci species, especially S. aureus, presumably due to a preference to non-biological materials, such as metal. Adherence is influenced by several factors, including the microenvironment, in which blood proteins from serum or plasma might influence adhesion and maybe biofilm formation. The aim of the present study was to test and to compare adherence of S. aureus and P. aeruginosa to different biological and non-biological surfaces in vitro. The question was addressed if coating of the surface by plasma or serum proteins influences bacterial adherence. METHODS: Adherence of radiolabeled bacteria to different surfaces in the presence or absence or serum/plasma proteins was measured over time. RESULTS: When testing adherence of S. aureus to plastic, titanium or to monolayers of epithelial cells (A549) or fibroblasts (Colo800) a clear-cut preference for non-biological surfaces, especially for titanium was seen. Using P. aeruginosa species a similar pattern without a significant difference was revealed. When mimicking the in vivo situation by pre-coating of titanium with human serum or plasma adherence was increased, especially when titanium was coated ("opsonized") by plasma. CONCLUSIONS: Bacterial adherence to surfaces is determined by a variety of factors such as temperature, the presence of nutrients, the absence of host defense systems and the configuration of the covered surface. In vivo, adherence to non-biological surfaces is also influenced by the microenvironment, especially plasma proteins, promoting biofilm formation.

Host defence against Staphylococcus aureus biofilms by polymorphonuclear neutrophils: oxygen radical production but not phagocytosis depends on opsonisation with immunoglobulin G.

Bacterial biofilms are increasingly recognised as a major cause of persistent infection and destructive inflammatory processes. In patients with biofilm infection, massive infiltration of leukocytes, particularly polymorphonuclear neutrophils is seen, and previous in vitro studies showed that PMN were able to phagocytose Staphylococcus aureus biofilms. We now addressed the question whether opsonisation of biofilms with immunoglobulin G and complement enhances the efficiency of phagocytosis, as it has been shown for "free-living" planktonic bacteria and other particulate materials. We found that incubation of biofilms with normal human serum resulted in IgG binding and in complement activation with deposits on the biofilm of C3bi. This "opsonisation", however, did not affect the adherence of PMN to the biofilms nor did it enhance degranulation or phagocytosis. The clearance of biofilms, however, was increased, and the oxygen radical production by the PMN depended critically on the coating of biofilms with IgG.

Destruction of bacterial biofilms by polymorphonuclear neutrophils: relative contribution of phagocytosis, DNA release, and degranulation.

Bacteria organized in biofilms are a common cause of relapsing or persistent infections, and the ultimate cause of implant-associated osteomyelitis. In these patients, biofilms of staphylococci are prevalent. Bacteria organized as biofilms are relatively resistant towards antibiotics and biocides, and it is also assumed that they may escape host defense mechanisms. In this context, we have studied how polymorphonuclear neutrophils (PMN), the "first line of defense" against bacterial infection, interact with biofilms generated in vitro. We found that PMN recognize biofilms and activate defense-associated reactions, including phagocytosis, degranulation of lactoferrin and elastase, and DNA release as well. Destruction of biofilms ensues, showing that biofilms are not inherently protected against the attack by phagocytic cells.

Activation of T Lymphocytes in Response to Persistent Bacterial Infection: Induction of CD11b and of Toll-Like Receptors on T Cells.

T cell activation is invariably associated with virus infections, but activation of T cells is also noted, for example, in patients with persistent bacterial infections with intracellular pathogens or localised bacterial biofilms. The latter is characterised by a destructive inflammatory process. Massive infiltration of leukocytes, predominantly of polymorphonuclear neutrophils (PMNs) and of T lymphocytes, is seen. While PMN influx into sites of bacterial infection is in line with their role as "first-line defence" a role of T cells in bacterial infection has not yet been delineated. We now found evidence for activation and expansion of peripheral blood T cells and an upregulation of Toll-like receptors 1, 2, and 4 on small portions of T cells. T cells recovered from the infected site were terminally differentiated and produced interferon gamma, a cytokine known to enhance functions of phagocytic cells, leading to the conclusion that infiltrated T cells support the local immuner defence.