Selective activation of helper and cytolytic T-cell functions of L3T4+ clones with either antireceptor antibody or phorbol ester and ionophore

After activation with specific antigen and antigen presenting cells (APC) L3T4+ inducer T-cell clones can lyse Ia+ APC. The present study characterizes the mechanism of activation and specificity of L3T4+ inducer cell-mediated cytolytic function. Two methods that bypass the physiological stimulus of antigen presented on Ia+ APC were used to activate L3T4+ clones. The first method utilized an antireceptor monoclonal antibody (MAb), KJ16.133, to activate KJ16.133+ clones. The activated clones expressed nonspecific cytolytic activity, killing target cells irrespective of their H-2 haplotype or their ability to express cell surface Ia molecules. The crosslinking of bound KJ16.133 antibody greatly enhanced cytolytic activity. This activation is receptor specific because KJ16.133- clones were not activated under identical conditions. The second method of activation was provided by a synergistic action of phorbol-12-myristate-13-acetate (PMA) and ionophore A23187. These agents nonspecifically activated all L3T4+ clones tested. The simultaneous presence of the two agents is required for maximal activation. Again, the activated clones expressed potent nonspecific cytolytic activity. These observations demonstrated that L3T4+ inducer T-cell-mediated killing can be separated into two stages: an activation step, which can be specifically and nonspecifically triggered and an effector phase which causes nonspecific lysis of bystander targets. The induction of nonspecific cytolytic activity by antireceptor MAb was inhibited by anti-L3T4 MAb (GK1.5). In contrast, activation of nonspecific cytolytic activity by treatment with PMA plus A23187 was not inhibited by anti-L3T4 MAb. Under the above activation conditions, antireceptor MAb selectively induced the secretion of IL-3 and expression of nonspecific cytolytic activity. However, there was little or no concomitant proliferation and production of IL-2. In contrast, activation by PMA plus A23187 coordinately induces expression of nonspecific cytolytic activity, secretion of lymphokines (IL-3 and IL-2), and cell proliferation. Thus, the anticlonotypic activation preferentially induces certain functions whereas activation with PMA plus A23187 is not selective.     

Histamine inhibits cell spreading and C3bi receptor clustering and diminishes hydrogen peroxide production by adherent human neutrophils.

Cell adherence plays a central role in many host defense mechanisms. Human peripheral blood neutrophils possess cell surface receptors that contribute to cell adherence or detachment. Receptors specific for the C3bi cleavage fragment of the third component of complement (CR3) promote adhesion, whereas histamine receptors promote detachment. In the present study, we tested the ability of histamine to down-regulate the physiological effects of CR3 receptors. Histamine decreased the binding of 51Cr-labeled neutrophils to complement-coated surfaces (C3-coated surfaces) in a dose-dependent fashion. Scanning electron microscopic and optical microscopic observations of neutrophils on C3-coated surfaces revealed polarized or spherical cell morphologies in the absence or presence of histamine, respectively. Histamine inhibited the ability of CR3 to cluster on plasma membranes of neutrophils adherent to C3-coated surfaces as shown by fluorescence microscopy. In addition, histamine diminished but did not abolish the FMLP-stimulated increase in plasma membrane CR3 expression as measured by fluorometry. Histamine did not inhibit the release of marker proteins from specific or gelatinase containing granules by neutrophils in suspension. Histamine also diminished the FMLP-stimulated production of respiratory burst oxidants from cells in suspension or cells allowed to adhere to fibrinogen substrates. We suggest that histamine may modulate selective changes in neutrophil function by diminishing adherence and preventing changes in cell shape following cell activation.     

Lectin binding to cystic stages of Taenia taeniaeformis

Studies of membrane glycoconjugates of Taenia taeniaeformis were initiated by assays of the lectin binding characteristics of 35-day-old cysticerci. Parasites fixed in glutaraldehyde were incubated with one of the following FITC-labelled lectins: Concanavalin A (Con A), Lens culinaris agglutinin (LCA), Ricinus communis agglutinin (RCA), peanut agglutinin (PNA), fucose binding protein (FBP) and wheat germ agglutinin (WGA) and either their specific or a nonspecific sugar. Ultraviolet microscopy revealed that only Con A and LCA bound in large amounts to the surface of cysticerci. This binding was partly inhibited by the specific sugar, but the nonspecific sugar had little effect. The lectin not removed by either of the sugars may have been bound nonspecifically to the charged glycocalyx. Lectins were primarily bound on the anterior third of the parasite around the scolex invagination. Kinetic studies of lectin interactions were carried out with LCA and RCA by spectrophotofluorometric analysis of the amount bound specifically or nonspecifically over a range of lectin concentrations. Lens culinaris lectin binding was found to be specific and involve 2 receptors which showed large differences in their affinity for lectin and prevalence on the surface. Ricinus communis lectin did not bind specifically but nonspecific interactions were observed. Adherence of small numbers of host cells was shown to have no measurable effect on the lectin binding characteristics. The results suggest that the major surface carbohydrates exposed are D-mannose and/or D-glucose residues with the other sugar groups poorly represented. This relatively homogeneous surface may have implications for the antigenicity of the parasite in its host.     

Role of the eosinophil in serum-mediated adherence of equine leukocytes to infective larvae of Strongylus vulgaris.

The adherence of equine leukocytes to Strongylus vulgaris infective larvae (L3) in the presence of normal and immune sera was examined in vitro. Immune sera promoted adherence of buffy coat cells from ponies with S. vulgaris-induced eosinophilia (eosinophilic ponies) to S. vulgaris L3. However, eosinophils in the buffy coat cells were the predominant adherent cell type. Studies using leukocyte populations enriched for eosinophils, neutrophils, and mononuclear cells from eosinophilic ponies support the observations using buffy coat cells that eosinophils were the main effector cells. Adherent eosinophils from eosinophilic ponies immobilized L3. Neutrophils were less adherent and did not immobilize L3. Mononuclear cells failed to adhere. Normal eosinophils from strongly-naive ponies did not immobilize S. vulgaris L3 in the presence of immune serum, suggesting the in vivo activation of eosinophils in eosinophilic animals. Immune serum promoted less adherence of buffy coat cells to Strongylus edentatus or mixed species of Cyathostominae L3, suggesting that the serum-mediated cellular adherence phenomenon was species-specific. Normal serum promoted less cellular adherence to S. vulgaris L3 than immune serum. The adherence mediated by normal serum was removed by heat inactivation, suggesting that this nonspecific phenomenon was a complement-mediated reaction. Immune globulins promoted reactions similar to that seen using heat-inactivated immune serum, whereas normal globulins did not promote adherence. Immune globulins absorbed with pieces of S. vulgaris adult worms did not promote the adherence of buffy coat cells to S. vulgaris L3, suggesting that adult and L3 stages share antigens important in this phenomenon that resulted in the removal of specific adherence antibody during absorption.     

Modulation of viral infections by cytokines.

The survival of a host challenged by viral infection depends on many factors. Some are specific, such as antiviral T-cell and B-cell responses. Others are nonspecific, such as intrinsic cellular resistance, macrophage activation, and activation of humoral protective mechanisms (e.g., complement, coagulation, etc.). Cytokines are important mediators and regulators of both types of host response. Furthermore, orchestration of specific humoral and cellular immune responses requires the participation of many cytokines. Details of the complex and overlapping roles that cytokines play in specific immune responses is beyond the scope of this review. Instead, this review will focus on the nonspecific antiviral effects of cytokines.     

Interaction of Listeria monocytogenes autolysin amidase with glycosaminoglycans promotes listerial adhesion to mouse hepatocytes

Adherence to the cell surface is a key event during infection of pathogenic microorganisms. We have previously reported that autolysin amidase (Ami) of Listeria monocytogenes promotes an efficient listerial adherence to mouse hepatocytes and requires for listerial pathogenicity. Cell wall anchoring (CWA) domain of Ami has been shown to bind lipoteichoic acid on listerial cell wall but the binding of Ami to host cell surface molecules remains to be determined. In this study, we present evidence here that Ami promotes efficient adherence of L. monocytogenes to mouse hepatocytes mediated by glycosaminoglycans (GAGs). The adhesion of L. monocytogenes wild type but not Ami-deficient mutant to the hepatocytes was dramatically attenuated by 4-nitrophenyl-β-D-xylopyranoside, a specific inhibitor of GAG association to cell surface. Full-length and truncated Ami were used to investigate the binding of Ami to GAGs and we found that four-repeated CWA of Ami is sufficient to bind GAGs on the host cell surface. Competitive assay and surface plasmon resonance demonstrated that Ami interacts with sulfated GAGs but not non-sulfated GAGs. The results suggest that Ami acts as an adhesin of L. monocytogenes to hepatocytes by interaction via its four-repeated CWA domain and sulfated GAGs.     

Membrane interactions in nerve myelin: II. Determination of surface charge from biochemical data.

In our accompanying paper (Inouye and Kirschner, 1988) we calculated the surface charge density at the extracellular surfaces in peripheral and central nervous system (PNS; CNS) myelins from observations on the dependency of the width of the extracellular space on pH and ionic strength. Here, we have determined the surface charge density of the membrane surfaces in myelin from its chemical composition and the localization of some of its molecular components. We then analyzed the attractive and repulsive forces between the apposed surfaces and calculated equilibrium periods for comparison with the measured values. The biochemical model accounts for the observed isoelectric range of the myelin period and, with the surface charge reduced (possibly by divalent cation binding or a space charge approximation), the model also accounts for the dependency of period on pH above the isoelectric range. At the extracellular (and cytoplasmic) surfaces the contribution of lipid (with pI approximately 2) to the net surface charge is about the same in both PNS and CNS myelin, whereas the contribution of protein depends on which ones are exposed at the two surfaces. The protein conformation and localization modulate the surface charge of the lipid, resulting in positively-charged cytoplasmic surfaces (pI approximately 9) and negatively-charged extracellular surfaces (pI approximately 2-4). The net negative charge at the extracellular surface is due in CNS myelin to lipid, and in PNS myelin to both lipid and (PO) glycoprotein. The net positive charge at the cytoplasmic surface is due in CNS myelin mostly to basic protein, and in PNS myelin to PO glycoprotein and basic protein. The invariance of the cytoplasmic packing may be due to specific short-range interactions. Our models demonstrate how the particular myelin proteins and their localization and conformation can account for the differences in inter-membrane interactions in CNS and PNS myelins.     

Electrostatic potential between surfaces bearing ionizable groups in ionic equilibrium with physiologic saline solution

In the past calculations of electrostatic properties of interacting cellular surfaces have been restricted by assumptions of fixed surface charge or surface potential. For the most part these calculations have been confined to a linear approximation and neglect the small but important complement of divalent cations in the cellular environment. In the present paper these limitations are removed. Solutions are obtained to the full non-linear Poisson-Boltzmann equation, treating the fraction of dissociated ionizable surface groups as a self-consistent functional of the electrostatic potential.     

Factors involved in adherence of lactobacilli to human Caco-2 cells.

A quantitative assay performed with bacterial cells labelled with [3H]thymidine was used to investigate factors involved in the adherence of human isolates Lactobacillus acidophilus BG2FO4 and NCFM/N2 and Lactobacillus gasseri ADH to human Caco-2 intestinal cells. For all three strains, adherence was concentration dependent, greater at acidic pH values, and significantly greater than adherence of a control dairy isolate, Lactobacillus delbrueckii subsp. bulgaricus 1489. Adherence of L. acidophilus BG2FO4 and NCFM/N2 was decreased by protease treatment of the bacterial cells, whereas adherence of L. gasseri ADH either was not affected or was enhanced by protease treatment. Putative surface layer proteins were identified on L. acidophilus BG2FO4 and NCFM/N2 cells but were not involved in adherence. Periodate oxidation of bacterial cell surface carbohydrates significantly reduced adherence of L. gasseri ADH, moderately reduced adherence of L. acidophilus BG2FO4, and had no effect on adherence of L. acidophilus NCFM/N2. These results indicate that Lactobacillus species adhere to human intestinal cells via mechanisms which involve different combinations of carbohydrate and protein factors on the bacterial cell surface. The involvement of a secreted bridging protein, which has been proposed as the primary mediator of adherence of L. acidophilus BG2FO4 in spent culture supernatant (M.-H. Coconnier, T. R. Klaenhammer, S. Kernéis, M.-F. Bernet, and A. L. Servin, Appl. Environ. Microbiol. 58:2034-2039, 1992), was not confirmed in this study. Rather, a pH effect on Caco-2 cells contributed significantly to the adherence of this strain in spent culture supernatant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a surface antigen on Loa loa microfilariae the recognition of which correlates with the amicrofilaremic state in man

Filarial infections induce a spectrum of disease in their natural hosts, and by correlating immunity found in individuals with their disease pattern, one may delineate non-pathogenic, protective mechanisms. Loa loa is causal of mild to moderate pathology, and it is unique among the human filaria in that adult worms are occasionally visible during subconjunctival migration. To study immune mechanisms controlling microfilaremia, sera from 15 subjects with amicrofilaremic occult loiasis (OL) were compared with sera from 10 subjects with microfilaremic loiasis (ML) microfilaremia, (greater than 4000/ml) for their reactions with living microfilariae (mf). An IFA was first used to detect antibodies able to bind to the surface of living L. loa mf. ML subjects either did not react (7/10) or reacted only very weakly (3/10). Highly reactive sera were found only in OL subjects; 7/15 gave very bright fluorescence, 5/15 gave moderate reactions, and 3/15 were negative. Most of these antibodies were of the IgG class. Sera from all subjects were also reacted with living mf in an antibody-dependent cellular adherence test using normal leukocytes. Sera that were strongly positive in IFA showed strong adherence and IFA-negative sera were non-reactive. To identify the Ag involved, mf were surface iodinated, detergent-extracted Ag were immunoprecipitated, and Mr was determined on SDS-PAGE. Several OL sera, all highly reactive in the above tests, precipitated a 23-kDa molecule with which all ML sea failed to react. Sera from a mandrill experimentally infected with L. loa also precipitated the 23-kDa Ag when taken post-patency. In conclusion, it appears that certain people who control L. loa microfilaremia have high levels of IgG antibodies that bind to a surface Ag of 23 kDa and are able to mediate cellular adherence.     

Caenorhabditis elegans: Stage Specific Differences in Cuticle Surface Carbohydrates

Stage-specific differences in wheat germ agglutinin (WGA) binding saccharides were demonstrated between the surfaces of the eggs, L1 larvae, young aduhs, and old adults of Caenorhabditis elegans. The WGA binding was to n-acetylglucosamine groups but not to terminally linked n-acetylneuraminic acids. An age-related decrease in WGA binding occurred in adults, supporting previous findings of a decrease in net negative cuticle surface charge during aging.     

Surface membrane proteins of Biomphalaria glabrata embryonic cells bind fucosyl determinants on the tegumental surface of Schistosoma mansoni primary sporocysts

Previous observations that in vitro adherence of Biomphalaria glabrata embryonic (Bge) cells to sporocyst larval stages of Schistosoma mansoni was strongly inhibited by fucoidan, a sulfated polymer of L-fucose, suggested a role for lectinlike Bge cell receptors in sporocyst binding interactions. In the present investigation, monoclonal antibodies with specificities to 3 major glycan determinants found on schistosomes, LacdiNAc, fucosylated LacdiNAc (LDNF), and the Lewis X antigen, were used in adhesion blocking studies to further analyze the molecular interactions at the host-parasite interface. Results showed that only the anti-LDNF antibody significantly reduced snail Bge cell adhesion to the surface of sporocysts, suggesting that fucosyl determinants may be important in larval-host cell interactions. Affinity chromatographic separation of fucosyl-reactive Bge cell proteins from fucoidan-bound Sepharose 4B revealed the presence of polypeptides ranging from 6 to 200 kDa after elution with fucoidan-containing buffer. Pre-elution of the Bge protein-bound affinity column with dextran (Dex) and dextran sulfate (DexS) before introduction of the fucoidan buffer served as controls for protein binding based on nonspecific sugar or negative charge interactions. A subset of polypeptides (approximately 35-150 kDa) released by fucoidan elution was identified as Bge surface membrane proteins, representing putative fucosyl-binding proteins. Far-western blot analysis also demonstrated binding reactivity between Bge cell and sporocyst tegumental proteins. The finding that several of these parasite-binding Bge cell proteins were also fucoidan-reactive suggests the possible involvement of these molecules in mediating cellular interactions with sporocyst tegumental carbohydrates. It is concluded that Bge cells have surface protein(s) that may be playing a role in facilitating host cell adhesion to the surface of schistosome primary sporocysts through larval fucosylated glycoconjugates.     

Attachment of Neisseria gonorrhoeae to the cellular pilus receptor CD46: identification of domains important for bacterial adherence

Pili of Neisseria gonorrhoeae mediate binding of the bacteria to human host cells. Membrane cofactor protein (MCP or CD46), a human cell-surface protein involved in regulation of complement activation, acts as a cellular pilus receptor. In this work, we examined which domains of CD46 mediate bacterial adherence. The CD46 expression was quantified and characterized in human epithelial cell lines. N. gonorrhoeae showed the highest adherence to ME180 cells, which have BC1 as the dominant phenotype. The BC isoforms of CD46 were expressed in all cell lines tested. The adherence was not enhanced by high expression of other isoforms, showing that the BC domain of CD46 is important in adherence of N. gonorrhoeae to human cells. To characterize the pilus-binding site within the CD46 molecule, a set of CD46–BC1 deletion constructs were transfected into COS-7 cells. Piliated N. gonorrhoeae attached well to CD46–BC1-expressing COS-7 cells. We show that the complement control protein repeat 3 (CCP-3) and the serine–threonine–proline (STP)-rich domain of CD46 are important for efficient adherence to host cells. Further, partial deletion of the cytoplasmic tail of CD46 results in low bacterial binding, indicating that the cytoplasmic tail takes part in the process of establishing a stable interaction between N. gonorrhoeae and host cells.     

Purification and partial characterization of platelet-derived adherence-inhibiting factor in guinea pig

Purification and partial characterization of adherence-inhibiting factor (AIF) of platelet-granule fraction in guinea pig were studied. When freshly prepared platelet-granule fraction was subjected to a gel filtration, two neutrophil adherence-inhibiting peaks, designated AIF-I (2,800 kDa) and AIF-II (12 kDa), appeared. AIF-I was sensitive to diisopropylfluorophosphate (DFP) and originated from lysosomes, whereas AIF-II was insensitive to DEP and localized in alpha-granules. Both AIFs were released from platelets by a thrombin stimulation. As the total activity of AIF-I was about 5-fold higher than that of AIF-II, AIF-I was purified and characterized. When purified AIF-I was analyzed on SDS-polyacrylamide gel electrophoresis, the 340 kDa protein band and the other large protein band were observed. Under reducing condition, AIF-I was separated into three components (340, 190 and 165 kDa). AIF-I significantly inhibited neutrophil adherence to artificial substrata and to type IV collagen-coated plastic surface, but not to fibronectin- or plasma-coated plastic surfaces, suggesting that AIF-I inhibits neutrophil adherence not only via nonspecific adsorption sites but also via type IV collagen receptors.     

Factors influencing adherence of Candida spp. to host tissues and plastic surfaces

Attachment of Candida spp. to host tissues and plastic surfaces is the first and a crucial step that initiates colonization by yeast cells and subsequent development of disseminated fungal infection. These infections are associated with high degree of morbidity, mortality and extra cost. Modern trends have focused not only on how best to treat but also on how to prevent Candida infections. To achieve this goal, the factors that influence the adherence of Candida spp. to biological and non biological surfaces have been studied. C. albicans adheres at a degree higher than that of the other Candida spp. and C. tropicalis adheres to a lesser extent. This may reflect the higher pathogenicity of C. albicans compared to the other Candida spp. Germinated C. albicans cells adhere to host tissue more readily than do yeast-phase. Sugars play an important role in the adherence of Candida spp. Overall, galactose was found to promote the adherence of Candida spp. to host tissues and plastic surfaces more than any other mono or disaccharide. Amino sugars on the other hand inhibit the adherence of the yeast cells. Divalent ions such as Ca2+ and Mg2+ promote the adherence of Candida spp. more than monovalent ions. Candida spp. express on their surface receptors, which interact with a wide variety of host proteins including fibrinogen, fibronectin, lamanin, and type I and IV collagen thus binding Candida spp. To glycoproteinaceous conditioning film at the blood-polymer interface. Coaggregation of Candida spp. with other bacteria promotes colonization of yeast cells to oral biofilm, host tissues, and to surfaces of the indwelling vascular catheters. These factors form the basis for the interference with the adherence of Candida spp.     

Central role of complement in passive protection by human IgG1 and IgG2 anti-pneumococcal antibodies in mice

Streptococcus pneumoniae is an important cause of morbitity and mortality worldwide. Capsule-specific IgG1 and IgG2 Abs are induced upon vaccination with polysaccharide-based vaccines that mediate host protection. We compared the protective capacity of human recombinant serogroup 6-specific IgG1 and IgG2 Abs in mice deficient for either leukocyte FcR or complement factors. Human IgG1 was found to interact with mouse leukocyte FcR in vitro, whereas human IgG2 did not. Both subclasses induced complement activation, resulting in C3c deposition on pneumococcal surfaces. Passive immunization of C57BL/6 mice with either subclass before intranasal challenge with serotype 6A induced similar degrees of protection. FcgammaRI- and III-deficient mice, as well as the combined FcgammaRI, II, and III knockout mice, were protected by passive immunization, indicating FcR not to be essential for protection. C1q or C2/factor B knockout mice, however, were not protected by passive immunization. Passively immunized C2/factor B(-/-) mice displayed higher bacteremic load than C1q(-/-) mice, supporting an important protective role of the alternative complement pathway. Spleens from wild-type and C1q(-/-) mice showed hyperemia and thrombotic vessel occlusion, as a result of septicemic shock. Notably, thrombus formation was absent in spleens of C2/factor B(-/-) mice, suggesting that the alternative complement pathway contributes to shock-induced intravascular coagulation. These studies demonstrate complement to play a central role in Ab-mediated protection against pneumococcal infection in vivo, as well as in bacteremia-associated thrombotic complications.     

Binding of Leishmania promastigotes to macrophages

Leishmania tropica promastigotes are easily attached to and engulfed by C3H peritoneal macrophages in vitro at 37 degrees C. Different sugars at 0.3-0.5 M inhibited in vitro the attachment of L. tropica promastigotes to C3H peritoneal macrophages with lactose (Gal-beta [1 leads to 4]Glc) being the most efficient. Inhibition of attachment is also affected by pre-treatment of promastigotes with galactose oxidase. Oligosaccharides extending from promastigote and amastigote cell surfaces contain an important proportion of non-reducing galactose as does the carbohydrate-rich factor (EF) excreted by promastigotes of L. tropica and L. donovani. This study suggests that Leishmania, an obligatory intracellular parasite, uses as a means of entering the host cell a cellular mechanism similar to that used in the removal of damaged cells from blood circulation. This mechanism is assumed to take advantage of the exposed sugars, particularly the exposed non-reducing galactose, on the parasite surface during the stage of attachment. Once the parasite is inside the cell, the EF it produces might have a protective function, being inhibitory to some of the host cell lysosomal enzymes.     

Leishmania major parasite stage-dependent host cell invasion and immune evasion

Leishmania pathogenesis is primarily studied using the disease-inducing promastigote stage of Leishmania major. Despite many efforts, all attempts so far have failed to culture the disease-relevant multiplying amastigote stage of L. major. Here, we established a stably growing axenic L. major amastigote culture system that was characterized genetically, morphologically, and by stage-specific DsRed protein expression. We found parasite stage-specific disease development in resistant C57BL/6 mice. Human neutrophils, as first host cells for promastigotes, do not take up amastigotes. In human macrophages, we observed an amastigote-specific complement receptor 3-mediated, endocytotic entry mechanism, whereas promastigotes are taken up by complement receptor 1-mediated phagocytosis. Promastigote infection of macrophages induced the inflammatory mediators TNF, CCL3, and CCL4, whereas amastigote infection was silent and resulted in significantly increased parasite numbers: from 7.1 ± 1.4 (after 3 h) to 20.1 ± 7.9 parasites/cell (after 96 h). Our study identifies Leishmania stage-specific disease development, host cell preference, entry mechanism, and immune evasion. Since the amastigote stage is the disease-propagating form found in the infected mammalian host, the newly developed L. major axenic cultures will serve as an important tool in better understanding the amastigote-driven immune response in leishmaniasis.     

Proteome analysis of human serum proteins adsorbed onto different titanium surfaces used in dental implants

Titanium dental implants are commonly used due to their biocompatibility and biochemical properties; blasted acid-etched Ti is used more frequently than smooth Ti surfaces. In this study, physico-chemical characterisation revealed important differences in roughness, chemical composition and hydrophilicity, but no differences were found in cellular in vitro studies (proliferation and mineralization). However, the deposition of proteins onto the implant surface might affect in vivo osseointegration. To test that hypothesis, protein layers formed on discs of both surface type after incubation with human serum were analysed. Using mass spectrometry (LC/MS/MS), 218 proteins were identified, 30 of which were associated with bone metabolism. Interestingly, Apo E, antithrombin and protein C adsorbed mostly onto blasted and acid-etched Ti, whereas the proteins of the complement system (C3) were found predominantly on smooth Ti surfaces. These results suggest that physico-chemical characteristics could be responsible for the differences observed in the adsorbed protein layer.     

Investigation of mechanisms involved in phagocytosis of Legionella pneumophila by human cells

Legionella pneumophila, the causative agent of Legionnaires' disease, is able to survive and multiply efficiently in a variety of mammalian cells. By using in vitro assays, the uptake of L. pneumophila into monocytes has shown to be mediated, at least in part, through attachment of complement-coated bacteria to complement receptors, but complement-independent phagocytosis could also be demonstrated. Since complement levels in the human lung are normally low, the role of complement-dependent phagocytosis in the pathogenesis of Legionnaires' disease is doubtful. However, the contribution of other potential phagocytosis-related host cell surface molecules to the phagocytosis of L. pneumophila has never been investigated. We therefore analyzed the role of complement receptors 1 (CD35) and 3 (CD11b/18), the lipopolysaccharide (LPS) receptor (CD14), the beta(1)-integrin chain of the fibronectin receptor (CD29), the intercellular adhesion molecule 1 (ICAM-1, CD54) and the transferrin receptor (CD71) in the complement-independent uptake of L. pneumophila. To exclude any influence of culture conditions onto phagocytosis rates, we compared a fresh clinical isolate with an agar-adapted isolate of L. pneumophila. In addition, we used three different host cell types (MM6, HeLa and Jurkat cells) expressing different rates of complement receptors. We could show that both strains of L. pneumophila were phagocytized by the three host cell lines to the same extent, but intracellular multiplication was only found in MM6 and, although to a much lesser degree, in Jurkat cells. Preincubation of MM6 cells with monoclonal antibodies directed against the above cited phagocytosis-related receptors did not result in inhibition of L. pneumophila uptake. We therefore conclude that typical phagocytosis-related cell surface receptors are not involved in the complement-independent phagocytosis of L. pneumophila.