Patent Application
20060292683
The invention relates to the field of biotechnology and microbiology, more specifically to antimicrobial medicines and antimicrobial immunogenic compositions.
Amyloid fibrils have been associated with pathology in a class of degenerated diseases like, for example, Alzheimer's disease and Creutzfeldt-Jakob. Amyloid structures also occur on the surface of microbial organisms like fungi and bacteria. The proteins in amyloid fibrils, oligomers, deposits and aggregates in degenerative diseases like Alzheimer's disease and Creutzfeldt-Jakob differ from those on the surface of bacteria and fungi with respect to amino-acid sequence and peptide length.
The amyloid-like structures are generally called hydrophobins on fungi, chaplins on gram-positive bacteria, and curli or tafi or aggregative fimbriae on gram-negative bacteria.
Generally, the virulence of a microorganism depends on characteristics like, for example, colonization factors, exo- and endotoxin production, replication time, resistance to antibiotics, and survival in the host macrophages, just to name a few. Methods to attenuate microorganisms for vaccination purposes were generally directed to the above-mentioned characteristics. Antimicrobial medicines are generally antibiotic compounds or bacteriostatic compounds.
Since resistance of microorganisms to antibiotic and bacteriostatic compounds is an ever-increasing problem, new methods for combating microorganisms are needed.
In certain aspects, the present invention provides at least one method or means that is suitable for at least in part combating a pathogenic microorganism or a microbial infection.
Disclosed herein is that microbial amyloid-like structures contain cross-β structure. Microbial hydrophobins, chaplins, tafis, curlis and aggregative fimbriae contain cross-β structure and are all capable of inducing protease activity in the body of a host. By inducing protease activity in a host tissue, a microorganism comprising cross-β, structure indirectly weakens the intercellular matrix thereby decreasing the integrity of the tissue. By this mechanism, the microorganism is able to invade the body.
Furthermore, the microbial cross-β structure is capable of binding intravascularly to factor XII. Binding of factor XII to the cross-β structure is followed by conversion of factor XII into a serine protease factor XIIa. The factor XIIa is involved in the release of bradykinin, which induces amongst other things, hemostasis and the release of more tissue type plasminogen activator (tPA) which is also a serine protease. Hence, microorganisms comprising cross-β structure, use the host cell's proteases to weaken the intracellular matrix of tissue and to cause intravascular hemostasis. Therefore, the presence of amyloid fibrils comprising cross-β structure renders a microorganism more virulent for a host.
The identification in the present invention of cross-β structure in the surface proteins of microorganisms now provides new methods for decreasing the virulence of a microorganism and offers new methods for inhibiting infection of a host by the microorganism.
A cross-β structure is a secondary/tertiary/quarternary structural element in peptides and proteins. A cross-β structure (also referred to as a “cross-beta” or a “crossbeta” structure or a “cbs”) is defined as a part of a protein or peptide, or a part of an assembly of peptides and/or proteins, which comprises an ordered group of β-strands, typically a group of β-strands arranged in a β-sheet, in particular a group of stacked β-sheets, also referred to as “amyloid.” A typical form of stacked β-sheets is in a fibril-like structure in which the β-sheets are stacked in either the direction of the axis of the fibril or perpendicular to the direction of the axis of the fibril. The direction of the stacking of the α-sheets in cross-β structures is perpendicular to the long fiber axis. A cross-β structure conformation is a signal that triggers a cascade of events that induces clearance and breakdown of the obsolete protein or peptide, a signaling pathway referred to as the “Cross-beta Pathway” for clearance of obsolete molecules and cells. When clearance is inadequate, unwanted proteins and/or peptides aggregate and form toxic structures ranging from soluble oligomers up to precipitating fibrils and amorphous plaques. Such cross-β structure conformation comprising aggregates underlie various diseases, such as, for instance, Huntington's disease, amyloidosis type disease, atherosclerosis, diabetes, bleeding, thrombosis, cancer, sepsis, inflammatory diseases, rheumatoid arthritis, transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease, Multiple Sclerosis, auto-immune diseases, diseases associated with loss of memory such as Alzheimer's disease, Parkinson's disease and other neuronal diseases (epilepsy), encephalopathy, encephalitis, cataract and systemic amyloidoses.
Cross-β structure is, for instance, formed during unfolding and refolding of proteins and peptides. Unfolding of peptides and proteins occur regularly within an organism. For instance, peptides and proteins often unfold and refold spontaneously during intracellular protein synthesis and/or during and/or at the end of their life cycle. Moreover, unfolding and/or refolding is induced by environmental factors such as, for instance, pH, glycation, oxidative stress, heat, irradiation, mechanical stress, proteolysis and so on. The terms unfolding, refolding and misfolding relate to the three-dimensional structure of a protein or peptide. Unfolding means that a protein or peptide loses at least part of its three-dimensional structure. The term refolding relates to the coiling back into some kind of three-dimensional structure. By refolding, a protein or peptide can regain its native configuration, or an incorrect refolding can occur. The term “incorrect refolding” refers to a situation when a three-dimensional structure other than a native configuration is formed. Incorrect refolding is also called misfolding. Unfolding and refolding of proteins and peptides involve the risk of cross-β structure formation. Formation of cross-β structure sometimes also occurs directly after protein synthesis, without a correctly folded protein intermediate.
Also disclosed is that a protease inhibitor is capable of decreasing the serine protease activity caused by cross-β structure. Therefore, the dissolving action on tissue and on blood clots is decreased, and the invasion of tissue by the microbial organism is at least in part decreased. Administration of the protease inhibitor as a medicine is very useful for treatment of an animal and/or a human being suffering from an infection with a microbial organism. Therefore, the present invention discloses the use of a protease inhibitor in the preparation of a medicament for the treatment of microbial infections. Preferably, the protease inhibitor is specific, i.e., the inhibitor essentially only inhibits, for example, serine protease factor XII.
A compound capable of binding to a cross-β structure also prevents the increase in protease activity and is, therefore, an inhibitor of a microbial infection. Therefore, the present invention also includes the use of a cross-β structure binding compound, like, for example, selected from those listed in Tables 3 through 5, in the preparation of a medicament for the treatment of microbial infections. Preferably, the compound comprises an antibody, or a functional fragment or derivative thereof, and/or a finger domain of a protease, specific for cross-β structure. An example of such a finger domain is described in Example 2 and in
Modern technology has enabled a skilled person to produce and select monoclonal antibodies. In this development also bi-specific antibodies are constructed. Generally a bi-specific antibody comprises two different binding specificities in single molecule. In, for example, a bi-specific antibody against cross-β structure of a microorganism, one antibody specificity is directed against the cross-β structure, while the other antibody specificity is preferably directed against another antigen of the microorganism. A bi-specific antibody has the advantage that the binding rate is increased because the antibody binds to more than one antigen. In one embodiment, a bi-specific antibody is generated which is directed against a particular (pathogenic) strain of microorganisms while leaving other (non-pathogenic) strains of the same microorganism untouched. Therefore, the present invention discloses the use according to the invention, wherein the antibody is a bi-specific antibody against a cross-62 structure and a microbial antigen.
In yet another preferred embodiment, the cross-β structure binding compound is any of the compounds mentioned in Tables 3 or 4 or 5 and, hence, the invention also discloses the use of a cross-β structure binding compound in the preparation of a medicament for the treatment of microbial infections, wherein the cross-β structure binding compound is selected from Tables 3 or 4 or 5.
It is clear to the skilled person that also combinations of any of the mentioned cross-β structure binding compounds are suitable for the treatment of microbial infections.
The above-described use of a protease inhibitor and/or cross-β structure binding compound (for example, an antibody and/or a bi-specific antibody or any of the compounds mentioned in Tables 3 or 4 or 5) is useful for the treatment of infections with microorganisms. Both gram -negative and gram-positive bacteria and also fungi have protein comprising cross-β structure on the surface. Therefore, the present invention discloses a use according to the invention, wherein the microorganism is cross-β structure-comprising Gram-positive microorganism. Because cross-β structure on a Gram-positive microorganism is generally present in a specific structure like a hydrophobin, or chaplin, the present invention discloses the use according to the invention, wherein cross-β structure comprises a hydrophobin or a chaplin.
Because a hydrophobin generally occurs on a fungus, the present invention discloses the use according to the invention, wherein the microorganism is a fungus. Hydrophobins have been detected in both the Ascomycetes and the Basidiomycetes. Both phyla represent most species in the fungal kingdom. Hydrophobins are generally expressed in dimorphic fungi. For the present invention, treatment of infections with pathogenic fungi is of preferred interest.
Because a chaplin generally occurs on a Gram-positive bacterium, the present invention discloses a use according to the invention, wherein the microorganism is a Gram-positive bacterium. In Gram-positive bacteria, chaplins are generally found on Streptomyces species and in other actinomycetes. Therefore, the invention provides a use, wherein the Gram-positive bacterium is an actinomycete or a streptomycete bacterium. Yet another example of a Gram-positive bacterium is a Staphylococcus, for example, a Staphylococcus aureus.
Because a curli or a tafi or a thin aggregative fimbria generally occurs on Gram-negative bacteria, the present invention discloses a use according to the invention, wherein the microorganism is a Gram-negative bacterium.
Important pathogens covered with curli or tafi or thin aggregative fimbriae are bacteria from the genera E. coli and Salmonella. Therefore, the present invention discloses a use according to the invention, wherein the Gram-negative bacterium is an E. coli bacterium or a Salmonella bacterium.
By disclosing the function of cross-β structure in the amyloid fibril for the virulence of a microorganism, the present invention discloses a method for decreasing the virulence of a microorganism by at least in part preventing the formation of cross-β structures. Decreasing is preferably achieved by deleting at least a part of a gene encoding an amyloid fibril forming protein. This results in at least partly decreased amyloid fiber formation on the surface and, therefore, decreased cross-β structure presence. The microorganism with decreased virulence is safer for a host and is useful as an immunogenic composition. Therefore, the present invention discloses a method for producing a less virulent microorganism, comprising deleting at least a part of a gene of the microorganism encoding an amyloid fibril forming protein. It is however also possible to make other mutations that result in less cross-β structure on the exterior of a microorganism. Such mutations include but are not limited to insertions, replacements etc.
Because part of the amyloid fibril is formed by cross-β structure, decreasing the amount of a cross-β structure-forming protein is a suitable way of decreasing the virulence of a microorganism without deleting other antigens. Such a microorganism preferably is more suitable for use in an immunogenic composition than a microorganism from which antigens have been stripped or altered in a process of attenuation. Therefore, the present invention discloses an immunogenic composition comprising a microorganism from which at least one gene, encoding a cross-β structure forming protein, has been at least in part deleted (or mutated in any other way, such that the amount of cross-β structure is at least in part decreased).
In a preferred embodiment of the invention, a protease inhibitor of the invention is used in combination with a fungicide compound. The fungicide preferably potentiates the effect of the protease inhibitor in decreasing an infection with a pathogenic fungus. A fungicide preferably is a compound generally known for its fungicidal activity. In this invention, for application to a host, (such as an animal or a human being), use of fungicidal compounds is restricted to those fungicides that are suitable for use on or in the host. Therefore, the present invention discloses a composition comprising a protease inhibitor and a fungicide.
In another preferred embodiment, a protease inhibitor of the invention is used in combination with a bactericide compound. The bactericide preferably potentiates the effect of the protease inhibitor in decreasing an infection with a pathogenic bacterium. In this invention, for application to a host (such as an animal or a human being), use of bactericidal compounds is restricted to those bactericides that are permitted for use on or in the host. Therefore, in a preferred embodiment, the present invention discloses a composition comprising a protease inhibitor and a bactericide.
Because cross-β structure induces serine proteases, inhibition of the serine proteases by specific serine protease inhibitors is preferred. Therefore, the present invention discloses a composition according to the invention, wherein the protease inhibitor is a serine protease inhibitor.
The invention further provides a composition comprising a protease inhibitor and a cross-β structure-binding compound. Such composition is particularly suitable for counteracting the microbial cross-β structure activity. The cross-β structure-binding compound preferably comprises an antibody, or a functional fragment or derivative thereof, and or a finger domain of a protease, specific for cross-β structure. In order to specifically counteract bacteria and/or fungi, the composition preferably further comprises a bactericide and/or a fungicide.
In yet another embodiment, the invention provides a (pharmaceutical) composition comprising a cross-β structure binding compound. In a preferred embodiment, the cross-β structure binding compound is selected from Tables 3 or 4 or 5. In yet an even more preferred embodiment, the compositions comprises at least two different cross-β structure binding compounds, which are preferably selected from Tables 3 or 4 or 5. In yet an even more preferred embodiment, the compositions comprise at least one or more of the cross-β structure binding compounds chaperones, soluble CD36 or soluble RAGE. In another embodiment, the composition further comprises at least one fungicide or at least a bactericide or a combination thereof.
In another embodiment, the invention provides a kit for detecting microbial contamination of a solution and/or a substance, the kit comprising a cross-β structure binding compound and a means for detecting binding of the cross-β structure to the binding compound. The means for detecting binding of cross-β structure to a cross-β structure-binding compound preferably comprises a tPA- and/or factor XII activation assay. In another preferred embodiment, the means for detecting binding of cross-β structure to a cross-β structure binding compound comprises visualization of a staining compound.
In yet another embodiment, the invention provide a method for detecting the presence of a microorganism in a solution and/or a substance, the method comprising providing a cross-β structure binding compound to the solution and/or substance and detecting whether any bound complex is present.
Examples of a suitable cross-β structure binding compound are, for example, outlined in Tables 3 or 4 or 5, and include a non-proteinaceous molecule, for example, a dye (Congo red or Thioflavin).
In a preferred embodiment, a cross-β structure binding compound is attached to a (solid) support or phase (i.e., is immobilized) such as, for example, on a sphere or a particle or a bead or a sheet or a strand of latex or agarose or glass or plastic or metal or any other suitable substance for immobilization of molecules. Such immobilization is especially useful when bound and unbound proteins must be separated. Depletion of a fluid from cross-beta structure and/or a protein comprising a cross-beta structure can be assessed, and/or enrichment of a solid support with bound cross-β structure binding compound with cross-beta structure and/or a protein comprising a cross-beta structure can be assessed, after contacting a fluid to a cross-β structure binding compound that is immobilized on a solid support. For example, a spike of a reference cross-beta structure can be applied to a tester sample and a control or reference sample. When contacting the samples with a cross-β structure binding compound the amount of cross-beta structure originally present in the sample will determine the amount of reference cross-beta structure that will bind to the cross-β structure binding compound. The differences in amount of reference cross-beta structure in a control sample and in a tester sample after contacting both samples to the cross-β structure binding compound can be assessed, for example, with a(n) (sandwich) ELISA specific for the reference cross-beta structure or, for example, by fluorescence measurement when a fluorescent label is coupled to the reference cross-beta structure. Alternatively, the amount of reference cross-beta structure bound to the cross-β structure binding compound can be assessed similarly. In yet an alternative approach, all proteins in a tester sample and in a reference or control sample can be labeled, for example, with biotin or a fluorescent label, prior to exposure to a cross-beta structure binding compound. The amount of labeled protein comprising cross-beta structure bound to the cross-β structure binding compound can subsequently be quantified and compared. In yet another approach, cross-beta structure that is bound to cross-β structure binding compound after contacting a reference sample and a tester sample with the cross-β structure binding compound, can be quantified after elution from the cross-β structure binding compound immobilized on a solid support, using a chromogenic assay. In the chromogenic assay, for example, dilution series of eluates of cross-β structure binding compound ligands are mixed with tissue-type plasminogen activator, plasminogen, a chromogenic substrate for plasmin and a suitable reaction buffer, and conversion of the substrate is followed in time upon 37° C. incubation.
Now that we have disclosed that a large number of microorganisms display cross-β structure on their exterior, this information is used to upgrade any solution, for example, a solution meant for human use. In one of the embodiments, the to be given solution is allowed to flow through a column in which matrix material is provided with immobilized cross-β structure binding compound (for example, one of the compounds as outlined in Tables 3 or 4 or 5). Any microorganism that is present in the solution and that comprises cross-β structure on its exterior will bind to the column and the solution is thus (at least in part) cleared from the microorganisms. Preferably, the conditions are such (for example, due to the presence of enough cross-β structure binding compound) that all microorganisms that comprise cross-β structure on their exterior are removed from the solution. In yet another embodiment, a cross-β structure binding compound is added to a solution and by binding of the binding compound to cross-β structure present on the exterior of a microorganism, the microorganism is subsequently not effective in provoking an adverse or unwanted or excessive reaction in a mammal that is treated with the solution. It is clear that in this embodiment, the cross-β structure binding compound may not be harmful to the mammal which subsequently receives the thus-treated solution. The invention thus also provides a method for at least in part removing a microorganism from a solution comprising contacting the solution with a cross-β structure binding compound. Preferably, the solution is subsequently used in the treatment of a mammal (preferably a human being). An example of such a solution is a dialysis solution.
The experimental part discloses that a cross-β structure binding compound is very useful in at least in part decreasing the pathogenicity of a microorganism and, hence, in yet another embodiment, the invention provides a method for decreasing the pathogenicity of a microorganism comprising providing the microorganism with a cross-β structure binding compound. With such a method cross-β structure present on the exterior of a microorganism is blocked or covered or shielded or neutralized and cannot participate in invading a mammal. Preferably, the microorganism is an E. coli or a Staphylococcus. The invention also provides the use of a cross-β structure binding compound for reducing the pathogenicity of a microorganism. Examples of suitable binding compounds are Thioflavin T (ThT), Congo red, intravenous immunoglobulins (IgIV), tissue-type plasminogen activator (tPA) or any of the other mentioned cross-β structure binding compounds of Tables 3 or 4 or 5.
The experimental part further discloses that interaction between an antigen-presenting cell and a pathogen is influenced by the presence or absence of a cross-β structure binding compound and, hence, the invention also provides a method for modifying the interaction between an antigen-presenting cell and a microorganism comprising changing the amount of a cross-β structure binding compound. It is clear that the microorganism is a microorganism that comprises cross-β structure on its exterior (for example, an E. coli or a Staphylococcus). Whether the amount of cross-β structure binding compound is increased or decreased depends on the specific situation. If one, for example, wants to stimulate the contact between a microorganism and an antigen-presenting cell, the amount of cross-β structure binding compound is reduced and if one wants to (at least in part) inhibit the contact between a microorganism and an antigen-presenting cell, the amount of cross-β structure binding compound is increased. An example in which one would like to increase the amount of cross-β structure binding compounds, is in patients with deleterious autoimmune disease, like, for example, multiple sclerosis (MS) or rheumatoid arthritis. These diseases are typically accompanied with all kinds of infection. By at least in part decreasing the interaction between a microorganism involved in the infection and an antigen-presenting cell, the human being suffering from MS or rheumatoid arthritis does not have to cope with the consequences of a microbial infection. In a preferred embodiment, the antigen-presenting cell is a dendritic cell.
The present invention refers at multiple locations to a microbial infection. It is clear to the skilled person that such different microbial infections may lead to a multitude of diseases. Examples of diseases that are preferably treated or prevented (i.e., prophylactic treatment) are mastitis or sepsis or any of the pathological conditions and diseases related to infection with any of the pathogens summarized in Table 6.
The invention further provides a method for in vitro determining whether a compound is capable of decreasing the pathogenicity of a microorganism comprising any of the methods as outlined in the experimental part herein. With such a method the efficiency of multiple cross-β structure binding compounds is easily determined and compared.
In yet another embodiment, the invention further provides one of the following methods:
(i) A method for at least in part inhibiting maturation of an antigen-presenting cell by a microorganism, comprising providing to the antigen-presenting cell and/or to the microorganism a cross-β structure binding compound.
In a preferred embodiment, the antigen-presenting cell is a dendritic cell.
(ii) A method for at least in part inhibiting induction of platelet activation by a microorganism, comprising providing to the platelet and/or to the microorganism a cross-β structure binding compound.
(iii) A method for at least in part inhibiting invasion of a microorganism into a host cell by extra-cellular matrix breakdown, comprising providing to the microorganism and/or the host cell a cross-β structure binding compound.
(iv) A method for at least in part reducing the vitality of a microorganism comprising providing to the microorganism a cross-β structure binding compound.
In a preferred embodiment, the microorganism is Gram-positive or a Gram-negative bacterium. In yet another preferred embodiment, the microorganism is a Streptomyces species or a Staphylococcus species or a Salmonella species or an E. coli or any of the pathogens mentioned in Table 6.
In another preferred embodiment, the microorganism is involved in the induction of sepsis and hence, the method is especially useful in the treatment of sepsis.
Any of the mentioned methods may be performed in vitro as well as in vivo and, hence, the invention also provides use of a cross-β structure binding compound in the preparation of a medicament in the treatment of a microbial infection.
The invention is further described with the use of the following illustrative examples.
Bacterial Cells with Amyloid-Like Core Protein Activate the Fibrinolytic System in Vitro
The Streptomyces coelicolor bacterium strain comprises a family of core proteins, chaplins A-H, which have adopted amyloid-like fibril conformation (Claessen et al., 2003). We now show that contacting the wild-type strain with tPA, plasminogen and plasmin substrate S-2251 results in activation of tPA and plasminogen (
The data show that the presence of the chaplin core proteins with amyloid-like conformation, on the surface of Streptomyces coelicolor cells activates the fibrinolytic pathway, by activation of tPA. Cells of a mutant strain lacking the genes that encode for the amyloid-like chaplin do not induce tPA activation. Activation of the fibrinolytic cascade or of factor XII, the key protein in the contact system of blood coagulation, by several different pathogens shows that in general core proteins are involved in interactions with the host haemostatic system. Our determination now of the F domain as the specific domain that interacts with protein aggregates (see Example 2) comprising amyloid-like cross-β structure conformation enables for tracing of pathogens with exposed amyloid-like proteins on their surface. The tPA- and factor XII activation assays are indicative in quality measures of solutions suspected for the presence of pathogenic infections.
tPA, Factor XII, Fibronectin and the Fibronectin Type I Domains of tPA, Factor XII and Fibronectin Bind to Protein Aggregates with Cross-β Structure Conformation
Previously, we established that tissue-type plasminogen activator specifically interacts with protein and peptide aggregates that comprise a cross-β structure conformation, a structural element found in amyloid-like polypeptide assemblies (Bouma et al., 2003; Kranenburg et al., 2002). Now, we expanded this analysis to other proteins that resemble tPA domain architecture and we separated domains of tPA. Binding of full-length tPA, factor XII and fibronectin, as well as of fibronectin type I (finger, F) domains of tPA and factor XII and F4-5 of fibronectin, to protein and peptide aggregates with cross-β structure conformation was analyzed in an ELISA. In
Cloning and Expression of Recombinant Fibronectin Type I Domains
Amino-acid sequences of recombinantly produced domains of tPA, fibronectin and factor XII, and the domain architecture of the recombinant constructs are depicted in
Alternatively, recombinant finger domains of fibronectin (F4-5) and tPA were expressed with a His-tag. Two fibronectin F4-5 constructs were cloned. One construct comprising the IgK signal sequence (vector 71, ABC-expression facility, Utrecht University/UMC Utrecht). With two designed primers (8, 9, see
The following part can roughly be divided into two parts. The first part describes criteria and means and methods in respect of the present invention and describes, for example, how to select and/or identify a pathogen which displays a cross-β structure comprising protein on its exterior, how to determine or verify whether a cross-β structure comprising protein is displayed. This part furthermore describes how to determine whether a cross-β structure is involved in pathogenicity and how to test whether, for example, the application of a cross-β structure binding compound results in reduced pathogenicity. In the second part some pathogens are subjected to the methods and means as described in the first part.
Role of cross-beta structure at the surface of pathogens in pathogenicity; leads for cross-beta structure binding compound-based therapies against infections
Pathogenicity of pathogens, such as bacteria, fungi, parasites and viruses, comes in several ways. The body reacts to pathogens with inflammatory responses and immunological responses. During infection components of the haemostatic system are also activated. It is disclosed herein that proteins with a cross-beta structure conformation at the surface of various pathogens mediate infection, including activation of components of the haemostatic system (Gebbink et al., 2005). A number of cell-based bioassays, blood enzyme activation tests and coagulation tests with a series of pathogens is conducted to provide evidence that compounds that interact with cross-beta structure are suitable to inhibit and/or prevent and/or counteract and/or abolish and/or reverse and/or diminish and/or interfere with infection and/or complications accompanied with or induced during infections. These examples have already provided (see below) and will further provide insight in the structure-function relationship of cross-beta structure during infection biology. The role of cross-beta structure is further assessed by including cross-beta structure binding compounds in the assays that may interfere with the pathogenic activity of the pathogens. Cross-beta structure binding compounds are used as potential inhibitory molecules in the in vitro (bio)assays and animal models. From these series of experiments it is concluded which cross-beta structure binding compounds act on cross-beta structure-mediated pathogenicity. The experiments provide leads for therapeutics for treatment of infections, based on cross-beta structure binding compounds.
A. Pathogens
The selection of pathogens that are analyzed for their pathogenic activity towards cells in our bioassays and for their influence on blood coagulation can be based on several criteria.
Some pathogens are known for their ability to bind and/or activate multiligand cross-beta structure binding proteins tissue-type plasminogen activator (tPA), factor XII and fibronectin (see Gebbink et al., 2005) and Tables 1 and 2 for pathogens that activate tPA or factor XII-mediated proteolytic processes). A series of observations in literature point to a role during infection for binding of cross-beta structure binding protein fibronectin to pathogens. For example, Spirochete Borrelia binding to subendothelial matrix was inhibited 48 to 63% by pretreatment of the matrix with anti-fibronectin antiserum. In addition, a 47 kDa fibronectin-binding protein expressed by Borrelia burgdorferi isolate B31 has been identified, and the cellular form of human fibronectin has been indicated as an adhesion target for the S fimbriae of meningitis-associated Escherichia coli. Moreover, the pavA gene of Streptococcus pneumoniae encodes a fibronectin-binding protein that is essential for virulence, and in Streptococcus pyogenes the gene of fibronectin-binding domain embp has been determined. Also for S. pyogenes several surface proteins are implicated in fibronectin binding, including protein F1, M and M-like. Curli fibers of Escherichia coli mediate internalization of bacteria by eukaryotic cells, and curli fibers bind fibronectin with high affinity. These binding and/or activation characteristics, together with binding studies performed with cross-beta structure binding dyes (Congo red, Thioflavin T) are considered to be a measure for the presence of proteins comprising cross-beta structure at the surface of the pathogens. As described before in this application, amyloid core proteins have been identified as being part of the core of several classes of pathogens. Those pathogens with cross-beta structure at their surface provide suitable models to analyze the role of cross-beta structure comprising proteins in the pathogenicity of these pathogens.
Alternatively, pathogen selection for our assays is driven by literature data showing pathogenicity of those pathogens towards cell types that are included in the assays, like, for example, cultured human umbilical vein endothelial cells (HUVEC), mouse microvascular bEnd.3 endothelial cells, THP-1 derived macrophages and/or monocytes, mouse bone marrow derived dendritic cells, human blood platelets and dendritic cells derived from human peripheral blood mononuclear cells. For example, for Staphylococcus aureus and Streptococcus pneumonia, both interaction with tPA and induction of platelet activation has been reported. Therefore, these pathogens provide suitable test cells for our analyses. For Staphylococcus aureus, binding of the fourth and fifth fibronectin type I domains of human fibronectin (Fn F4-5) has been demonstrated. Fn F4-5 are the domains that interact with a fibrin network and that bind to cross-beta structure in general.
Finally, selection criteria for pathogens that can be included in our further studies to unravel the role of pathogen surface proteins with cross-beta structure in host invasion, infection, immunity, inflammation and hemostasis is based on literature data describing the interaction of multiligand cross-beta structure receptors of a host with pathogens. For example, CD36 is an essential receptor on macrophages that mediates internalization of pathogens, for example, Staphylococcus aureus, and triggers signaling pathways resulting in tumor necrosis factor-α (TNF-α) and interleukin-12 (IL-12) expression. This makes Staphylococcus aureus an attractive model pathogen to study whether S. aureus core proteins with cross-beta structure are involved in the interaction with, e.g., CD36. For a first series of experiments, for example, a Streptococcus pneumonia strain is selected. Furthermore, for example, a Gram-positive bacterium is included in the studies, for example, a Staphylococcus strain is used, for example, a Staphylococcus aureus strain, for example, a Staphylococcus aureus Newman strain. In addition, a Gram-negative bacterium is included in the studies, for example, an Escherichia coli strain is used, for example, Escherichia coli strain TOP10 (Invitrogen, 44-0301) or, for example, Escherichia coli strain MC4100.
It is, therefore, clear for a skilled person that a suitable pathogen (i.e., a pathogen comprising cross-β structure on its exterior) may be selected in a variety of ways of which some have been non-limiting mentioned above.
B. Determination of the Presence of Cross-Beta Structure Protein Conformation on Pathogens
With the selected pathogens (see selection criteria above) a series of analyses is performed that provide insight in the presence of cross-beta structure protein conformation. Standard Congo red and Thioflavin T binding and fluorescence assays are conducted. For this, pathogens are incubated with the amyloid binding dyes and fluorescence enhancement is determined. Alternatively, pathogens are fixed, stained with the dyes and binding is analyzed under a fluorescence microscope or under a direct light microscope using polarized light (Congo red birefringence), or free pathogens are incubated with dyes and fixed afterwards, before microscopic analysis. Presence of cross-beta structure on pathogens may also be assessed by culturing pathogens on culture plates containing Congo red or Thioflavin T or Thioflavin S. Presence of cross-beta structure can be determined by visual inspection. Pathogen cells can also be analyzed by using electron microscopy, to determine the presence of fibrillar structures at the pathogen surface. Interaction with cross-beta structure binding proteins tissue-type plasminogen activator and factor XII is assessed using chromogenic assays. For this purpose, concentration series of the pathogens are mixed with 100-1000 pM tPA, 5-200 μg/ml plasminogen and 0.1-1 mM chromogenic plasmin substrate S2251 (Chromogenix), and conversion of plasminogen to plasmin upon tPA activation by cross-beta structure is followed in time during 37° C.-incubation. For factor XII activity measurement, concentration series of pathogen are mixed with 0.1-50 μg/ml factor XII, 0-5 μg/ml prekallikrein, 0-5 μg/ml high molecular weight kininogen and either chromogenic factor XII substrate S2222 (Chromogenix) for direct measurement of factor XII activity, or chromogenic kallikrein substrate Chromozym-PK (Boehringer-Mannheim) for indirect factor XII activity, and substrate conversion is followed in time spectrophotometrically during 37° C. incubation. Alternative to the tPA and factor XII activation assays, presence of cross-beta structure on pathogens may also be assessed using ELISA set-ups. For this purpose, for example, pathogens are immobilized onto the wells of ELISA plates. Subsequently, plates are blocked with a blocking solution, and concentration series of cross-beta structure binding compounds are added to the wells. Binding of the cross-beta structure binding compounds is determined using specific antibodies. Examples of cross-beta structure binding compounds that may be used for this approach are tPA, factor XII, fibronectin, finger domains derived from tPA, factor XII, fibronectin or hepatocyte growth factor activator (HGFA), soluble fragment of receptor for advanced glycation end products (sRAGE), soluble extracellular fragments of low density lipoprotein receptor related protein (LRP cluster 2, LRP cluster 4), (hybridoma) antibodies, intravenous immunoglobulins (IgIV or IVIg, either or not a fraction that is enriched by applying a cross-beta structure affinity column), or chaperones like, for example, BiP, HSP70, HSP90. All of the above-listed analyses are preferably performed with solutions before and after centrifugation for one hour at 100,000*g, or preferably before and after filtration using a 0.2 μm filter. Positive controls that are preferably included in the assays are glycated hemoglobin, amyloid-β and amyloid γ-globulins, prepared by incubation of β-globulins in H2O at 37° C., after dissolving lypohilized γ-globulins in 1,1,1,3,3,3-hexafluoro-2-propanol and trifluoro acetic acid, followed by air-drying.
C. Cell Assays and Other Bioassays
1. General Overview of the Use of Bioassays
In all of the cell-based bioassays described below, cell concentration series of the selected pathogens (see above) are added to cultured cells to determine the optimal concentration for subsequent inhibitory studies. When suitable pathogen cell densities are determined, that induce a pathogenic response in the cultured cells, concentration series of putative blockers of the obtained pathogenic effects are tested by premixing concentrations of pathogen cells with concentration series of cross-beta structure binding compounds, and then by adding the premixes to cultured cells, followed by standard read-out measurements for pathogenicity. Alternatively, pathogens are first pelleted, solution with residual cross-beta structure binding compound is discarded, the pathogen cell pellet is resuspended in buffer and washed before applying the pathogen to the bioassay. In this way, information is obtained about the role of pathogen proteins comprising cross-beta structure in inducing pathogenic conditions in the cell cultures. Positive controls that are preferably included in the bioassays are glycated hemoglobin, amyloid-β, CpG, apoptotic cells of any kind, necrotic cells of any kind and lipopolysaccharide.
2. In Vitro Murine Dendritic Cell Assay
Immunity against pathogens is dependent on the presentation of antigens by antigen-presenting cells (APC), such as dendritic cells. Cultured murine dendritic cells (DCs) are applied as a model for immunogenicity of cross-beta structure bearing pathogens. For this purpose, DCs are isolated from the hind legs of, for example, 8 to 12-week-old Black-6 mice. Bones are isolated and rinsed in 70% ethanol, rinsed in RPMI-1640 medium with 25 mM HEPES, with 10% fetal calf serum, penicillin and Streptomycin. Then the bones are flushed with this buffer, in both directions. Eluates are cleared from erythrocytes by adding erythrocyte-specific lysis buffer (for example, obtained from the local UMC Utrecht Pharmacy Dept., catalogue number 97932329). Eluates are analyzed for viable cells by culturing them in cell culture plates. At this stage, the medium is enriched with 10 ng/ml GM-CSF. DCs grow in suspension or on a layer of macrophage cells. Using a FACS and specific antibodies, it is determined whether DCs are present and the activation state is analyzed. Preferably, the levels of cell surface receptors involved in endocytosis and co-stimulatory molecules, such as B7.1, B7.2, MHC class II, CD40, CD80, CD86 is determined on preferably CD11c-positive cells. Alternatively, activation of NF-κB and/or expression of cytokines will be used as indicators of activation of cells involved in immunogenicity, such as APC and DC. Preferably, the following cytokines are quantified: TNFα, IL-1, IL-2, IL-6, and/or IFNγ. Preferably, the cytokine levels are quantified by ELISA. Alternatively, the mRNA levels are quantified. For a person skilled in the art it is evident that function of APC and DC are tested as well.
Alternatively, a stable DC line or other antigen-presenting cells is used to test beneficial effects of depletion or neutralization of misfolded proteins with cross-beta structure on pathogens (Citterio et al., 1999).
3. In Vitro Generation of Peripheral Blood Human Monocyte-Derived Dendritic Cells and Activation Assay
Human DCs are generated from non-proliferating precursors selected from peripheral blood mononuclear cells (PBMCs), essentially by published methods (Sallusto and Lanzavechhia, 1994). In brief, the hematocrit fraction of freshly drawn citrated human blood or of buffy coat blood is used. Using the Ficoll/Lymphoprep-based separation-centrifugation method, PBMCs are separated. Subsequently, monocytes are purified from this PBMC fraction by using the Percoll-based separation-centrifugation and adherence method. The CD14-positive monocytes (0.5×106/ml) are cultured for approximately six to seven days (37° C., 5% CO2) in serum-free medium enriched with, for example, 10 ng/ml GM-CSF and 10 ng/ml IL-4. Presence of immature DCs is, for example, determined by Fluorescence Activated Cell Sorting (FACS) analysis for the presence or absence of CD14, CD1a, CD80, CD40, CD86, HLA-DR, CD83, CD206, CD36 and CD163 surface expression. Relative abundant presence of CD1a, CD36, CD40, CD86 and CD206 and relative low content of CD14-positive, CD80-positive, CD83-positive and CD163-positive cells will serve as a quality measure for the immature DCs. After obtaining the immature DCs upon stimulation with GM-CSF and IL-4, cells are, for example, be incubated for 16 to 72 hours with a concentration series of cultured Streptococcus pneumoniae, Escherichia coli or Staphylococcus aureus Newman in PBS or in buffer comprising cross-beta structure binding compounds like, for example, Congo red, Thioflavin T, tPA, finger domains and IgIV. To determine the influence of the bacterial cells on the DCs, typically surface density of CD83, CD86, CD80, CD163, CD14, CD40, CD36, scavenger receptor A, LRP, CD1a, HLA-DR, LOX-1, Toll-like receptor-2 (TLR2), TLR4, TLR9 and mannose receptor/CD206 and/or the percentage of positive cells with respect to the DC surface molecules are, for example, measured using FACS.
4. In Vitro Human Umbilical Vein Endothelial Cell and Murine Microvascular bEnd.3 Endothelial Cell Activation Assay
Glycated proteins comprising a cross-beta structure and amyloid-β induce inflammatory responses and are believed to contribute to the pathogenesis of certain protein misfolding diseases (diabetes type II, Alzheimer's disease). In general, misfolded proteins induce cellular dysfunction with enhanced expression or activation of inflammatory signals. The effect of misfolded proteins on endothelial cell (dys)function is, for example, measured by determining the levels of reactive oxygen species or nitric oxide or tissue factor in response to misfolded proteins. Human umbilical vein endothelial cells (HUVEC) that are isolated and cultured, according to standard protocols, are used, or other endothelial cells such as the murine microvascular bEnd.3 endothelial cell line. The levels of reactive oxygen species (ROS), like, for example, nitric oxide, are monitored using fluorescent probes, such as CM-H2DCF-DA. Alternatively, cell viability is monitored by standard MTT-assay. The levels of tissue factor expression is determined using a chromogenic assay with purified factor VII, purified factor X and chromogenic substrate S-2765 (Chromogenix), using cell lysates. For example, bEnd.3 cells are seeded at 120,000 cells/well of a 96-well culture dish, and cultured overnight. Cells are subsequently exposed to a dilution series of overnight cultured E. coli cells or S. aureus cells, which are resuspended in PBS after centrifugation and discarding the Luria broth supernatant, or which are resuspended in a solution comprising cross-beta structure binding compounds Thioflavin T (Sigma-Aldrich), Congo red (Sigma-Aldrich), tissue-type plasminogen activator (Actilyse, Boehringer-Ingelheim) and intravenous immunoglobulins (IgIV, Octagam, Octapharma). Changes in levels of ROS are followed in time during a one-hour incubation at 37° C., at, for example, two-minute intervals.
The cultured primary cells and the cell line provide the opportunity to perform in vitro cell assays that are accepted in research community as model systems for certain disease states.
5. Phagocytosis of Cross-Beta Structure Comprising Pathogens
The uptake of cross-beta structure comprising pathogens, and the effect of cross-beta structure binding compounds are studied in vitro using cultured cells, preferably monocytes, dendritic cells, or macrophages or similar cells, for example, U937 or THP-1 cells. Preferably, cross-beta structure comprising pathogens are labeled, preferably with 125I or a fluorescent label, preferably FITC, covalently attached to the molecule by a linker molecule, preferably ULS (universal Linkage system) or by applying an alternative coupling method. Cells are preferably labeled with mepacrin or other fluorescent labels, such as rhodamine. Phagocytic cells are incubated in the presence of labeled cross-beta structure comprising cells in the presence or absence of a cross-beta structure binding compound (see below). After incubation, preferably during several hours, the uptake of labeled molecules or cells is measured preferably using a scintillation counter (for 125I) or by FACS-analysis (with fluorescent probes) or immunofluorescent microscopy. The uptake of pathogen cells is also counted under a light microscope with visual staining of these cells.
Alternatively, the response of cells that are involved in phagocytosis to cross-beta structure comprising pathogens is also assessed by measuring expression levels of several markers for an inflammatory/activation/thrombogenic response. Using commercially available ELISAs, expression levels of tumor necrosis factor-α and interleukin-8 are determined upon exposure of, for example, macrophages to cross-beta structure comprising pathogens. Expression levels of tissue factor are determined using a chromogenic assay with chromogenic substrate S2765 (Chromogenix), factor VII and factor X.
6. Ex vivo Human Blood Platelet Aggregation Assay
The influence of cross-beta structure binding compounds on blood platelet aggregation induced by cross-beta structure comprising pathogens is tested with washed platelets in an aggregometric assay. Freshly drawn human aspirin free blood is mixed gently with citrate buffer to avoid coagulation. Blood is spinned for 15 minutes at 150*g at 20° C. and supernatant is collected; platelet rich plasma (PRP). Buffer with 2.5% trisodium citrate, 1.5% citric acid and 2% glucose, pH 6.5 is added to a final volume ratio of 1:10 (buffer-PRP). After spinning down the platelets upon centrifugation for 15 minutes at 330*g at 20° C., the pellet is resuspended in HEPES-Tyrode buffer pH 6.5. Prostacyclin is added to a final concentration of 10 ng/ml, and the solution is centrifuged for 15 minutes at 330*g at 20° C., with a soft brake. The pellet is resuspended in HEPES-Tyrode buffer pH 7.2 in a way that the final platelet number is adjusted to 200,000 to 250,000 platelets/μl. Platelet counts are adjusted to approximately 300,000 platelet/μl when PRP is used. Platelets are kept at 37° C. for at least 30 minutes, before use in the assays, to ensure that they are in the resting state. Platelets of approximately five donors are isolated separately.
For the aggregometric assays, platelet solution is added to a glass tube and prewarmed to 37° C. A stirring magnet is added and rotation is set to 900 rpm, and the apparatus (Whole-blood aggregometer, Chrono-log, Havertown, Pa., USA) is blanked. A final volume of 1/10 of the volume of the platelet suspension is added (typically 300 μl to 300 μl platelet suspension), containing the agonist of interest and/or the premixed antagonist of interest, prediluted in HEPES-Tyrode buffer pH 7.2. Alternatively, pathogens are first pelleted, solution with residual cross-beta structure binding compound is discarded, the pathogen cell pellet resuspended in buffer and washed before applying the pathogen to the bioassay. Aggregation is followed in time by measuring the absorbance of the solution that will decrease in time upon platelet aggregation. As a positive control, either 10 μg/ml collagen (Kollagenreagens Horm, NYCOMED Pharma GmbH, Linz, Austria; lot 502940), or 5 μM of synthetic thrombin receptor activating compound TRAP, or 10-100 μg/ml glycated hemoglobin or 10-100 μg/ml amyloid-β is used. Aggregation is recorded for at least 15 minutes.
7. Ex Vivo Human Plasma Coagulation Assays
For analysis of the influence of cross-beta structure comprising pathogens on the characteristics of blood coagulation, and for analysis of the effects of cross-beta structure binding compounds on the influence of pathogens on coagulation, two standard coagulation tests are performed on, for example, a KC10 Coagulometer. Pooled human plasma of approximately 40 apparently healthy donors is clotted by adding either negatively charged phospholipids, CaCl2 and kaolin when an activated partial thromboplastin time (aPTT) is considered, or tissue factor rich thromboplastin and CaCl2 when prothrombin time (PT) determinations are considered. APTTs and PTs are performed as follows. Plasma is incubated with concentration series of pathogen for, for example, 15 minutes to 120 minutes at room temperature or at 37° C. Pathogen cells are pelleted by centrifugation, for example, for 30 seconds with 16,000*g, and plasma supernatant is subsequently applied in either an APTT or a PT. At conditions that influence the coagulation tests, preincubations of pathogens with concentration series of cross-beta structure binding compounds are performed, before applying the pathogens to plasma, or in an alternative way, pathogens and cross-beta structure binding compounds are applied to plasma together. For an APTT analysis, 50 μl of plasma is mixed with 50 μl of a physiological buffer. Next 25 μl of 900 μg/ml Kaolinum Ponderosum (Genfarma) and 120 μM lipid vesicles (phosphatidyl serine/phosphatidyl choline/phosphatidyl ethanolamine) in a 20/40/40% (v/v) ratio are added, and the mixture is prewarmed to 37° C. To start the assay, 25 μl of a 50 mM CaCl2 solution is added. For a PT analysis, 50 μl of (pretreated) plasma is combined with 50 μl H2O and is incubated for five minutes at 37° C. The PT analysis is started by adding 50 μl of a Thromborel S stock, which is prepared at twice the concentration as recommended by the manufacturer (DADE Behring).
8. In Vitro Murine Monocyte Tissue Factor, Tumor Necrosis Factor-α and Interleukin-8 Expression
THP-1 cells are cultured using conditions that provide monocytes. For this purpose we culture the cells in Iscove's Modified Dulbecco's Medium (IMDM) with 5% fetal calf serum and 50 μg/ml gentamycin. For further studies THP-1 cells are also stimulated and differentiated by exposing the cells to, for example, phorbol 12-myristate 13-acetate (PMA) and/or Tetra-Phorbol-Acetate (TPA) and/or interferon-γ and/or lipopolysaccharides.
Analysis of Tissue Factor Expression by THP-1
For tissue factor expression analysis purposes, THP-1 cells are cultured in IMDM without gentamycin and streptomycin. At day 0, one ml of cells is seeded at 1×106 cells/ml in the wells of six-well culture plates. At day 1, cells are stimulated with putative agonists and/or antagonists for six hours at 37° C. (regular culturing conditions). Positive control is a concentration series of LPS, negative control is buffer. Agonists that are tested are misfolded proteins comprising cross-beta structure and pathogens comprising amyloid core proteins with cross-beta structure and apoptotic cells and necrotic cells. Antagonists that are tested are inhibitory antibodies against THP-1 surface receptors involved in signal transduction upon exposure to cross-beta structure, like, for example, antibodies against CD91/LRP, CD36, receptor for advanced glycation end products (RAGE), scavenger receptor A, scavenger receptor B-I, Toll-like receptor 4. Other antagonists that are included for analysis of inhibitory properties are cross-beta structure binding compounds like, for example, Congo red, Thioflavin T, tPA, fibronectin, BiP, HSP60, HSP70, HSP90, gp96, soluble fragments of LRP (cluster 2, cluster 4), soluble fragment of RAGE, finger domains, antibodies, IgIV. After stimulation, cells are pelleted by five minutes centrifugation at low speed. Supernatants are analyzed for TNF-α and/or IL-8 levels, using commercially available ELISAs. The cell pellet is resuspended in 100 μl TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.0-7.3). Next, the cells are frozen and thawed for four subsequent cycles. Cells are centrifuged for ten minutes at 16,000*g and the supernatant is used for further analysis. First, protein concentrations are determined using a regular protein concentration assay like, for example, a Bicinchoninic Acid (BCA) Protein Assay. Protein concentrations are equalized between samples with TBS to correct for variations in cell density. For analysis of tissue factor levels, 50 μl of cell lysate is incubated with 50 μl buffer comprising 10 μg/ml factor X, 5 U/ml factor VII (FVII) and 5 mM CaCl2, for 45 minutes at 37° C. in wells of a 96-well plate. Then, 50 μl of a 4.5 mM chromogenic activated factor X substrate S2765 (Chromogenix) stock is added. Conversion of the substrate by activated factor X at 37° C. is recorded in time for at least ten minutes, by absorbance readings at 405 mm.
D. Cross-Beta Structure Binding Compounds Used as Potential Inhibitors of Cross-Beta Structure-Mediated Pathogenicity of Pathogens
To be able to analyze the role of cross-beta structure comprising proteins on pathogens in pathogenicity, as assessed in the above-listed series of bioassays, a series of cross-beta structure binding compounds is included in the assays as potential inhibitors of cross-beta structure-mediated pathogenicity. Cross-beta structure binding compounds are typically included in the assays at concentrations of 1-5000 μg/ml, or 1 nM —1 mM. Examples of cross-beta structure binding compounds that are used are Congo red, Thioflavin T, Thioflavin S, tPA, factor XII, fibronectin, finger domains derived from tPA, factor XII, fibronectin or HGFA, sRAGE, sLRP, LRP cluster 2, LRP cluster 4, (hybridoma) antibodies, IgIV (either or not a fraction that is enriched by applying a cross-beta structure affinity column), soluble extracellular fragment of LOX-1, soluble extracellular fragment of CD40, soluble extracellular fragment of CD36, or molecular chaperones like, for example, clusterin, haptoglobin, BiP/grp78, HSP60, HSP70, HSP90, gp96 (see Tables 3 through 5 for more examples of cross-beta structure binding compounds).
A. Culturing of Staphylococcus aureus Newman and Escherichia coli TOP10, and Preparing Reference and Tester Cell Samples
Staphylococcus aureus Newman, which was a kind gift of Dr. Jos van Strijp and Dr Kok van Kessel (Dept. of Microbiology, University Medical Center Utrecht, the Netherlands) was plated on a blood plate from a stock stored at −70° C., and incubated overnight at 37° C. The plate was stored at 4° C. Escherichia coli strain TOP10 (Invitrogen, 44-0301) was plated on agar with Luria broth medium from a −80° C. glycerol stock, and incubated overnight at 37° C. The plate was stored at 4° C. Overnight cultures of 5 ml in Luria broth medium were grown at 37° C. with vigorous shaking and aeration, by streaking a single colony with the tip of a pipet and transferring the tip to the medium in a 15-ml tube. The cell density in overnight cultures was determined by measuring the absorbance at 600 nm (A600=1 is equivalent with 0.8×109 cells/ml). Cells were pelleted by centrifugation for one minute at 16,000*g or for ten minutes at 3,000*g. Medium was discarded. One half of the cells were resuspended in PBS in 1/10 of the original medium volume (10× concentration of the cells) by pipetting and swirling. These cells were used as reference cells. The second half of the cells was designated as “tester” cells and was resuspended in PBS with 5 mM Thioflavin T (again 1/10 of the original medium volume), by pipetting and swirling, as with all subsequent handlings. The tester cells were incubated for five minutes at room temperature with constant swirling. Cells were pelleted by centrifugation for 30 seconds at 16,000*g and supernatant was discarded. Cells were resuspended in 5 mM Congo red in PBS and incubated in a way similar to the Thioflavin T incubation. After pelleting the cells, they were resuspended in PBS and again pelleted by centrifugation for 30 seconds at 16,000*g. Supernatant was discarded. Finally, tester cells were resuspended in a solution of 25 μM tissue-type plasminogen activator (Actilyse, Boehringer-Ingelheim) and 25 mg/ml intravenous immunoglobulins (IgIV, Octagam, Octapharma), in approximately 1/150 of the original medium volume (75× concentration of the cells). After a 30-minute incubation, cells were pelleted, resuspended in PBS (approximately 1/10 of the original medium volume) and kept at room temperature for use at the same day or kept at 4° C. for later use within 72 hours E. coli cell suspension was yellow-light orange after all subsequent incubations, S. aureus cell suspension was red. Initial cell densities of the overnight cultures were 1.8×109 cells/ml for the S. aureus and 1.3×109 for the E. coli cells. The work suspensions had cell densities of 1.8×1010 cells/ml and 1.8×1010 cells/ml, respectively.
Reference and tester Staphylococcus aureus Newman cells or Escherichia coli TOP10 cells obtained as described above were applied in a series of bioassays. Cells at various indicated densities were analyzed for their activity in the assays C-3 (activation of human DCs), C-4b (ROS production in murine ECs), C-6 (platelet aggregation), C-7 (plasma coagulation, PT and aPTT analyses) and C-8 (tissue factor expression in THP-1 monocytes), as described above. In addition, plasmin generation in a chromogenic tPA/plasminogen activation assay will be assessed and activation of factor XII and prekallikrein will be assessed in a chromogenic assay. For this purpose dilution series of the pathogen cells will either be mixed with final concentrations of 400 pM tPA, 0.2 μM plasminogen (purified from human plasma) and 0.8 mM chromogenic plasmin substrate S2251 (Chromogenix) or chromogenic plasmin substrate Biopep-1751 (Biopep, France) in a physiological buffer, or with 0.3 mM chromogenic kallikrein substrate Chromozym-PK (Roche Diagnostics, Almere, The Netherlands), 1 μg/ml zymogen factor XII (#233490, Calbiochem, EMD Biosciences, Inc., San Diego, Calif.), human plasma prekallikrein (#529583, Calbiochem) and human plasma cofactor high-molecular weight kininogen (#422686, Calbiochem). For the factor XII assay, the assay buffer contained HBS (10 mM HEPES, 4 mM KCl, 137 mM NaCl, pH 7.2). Plasmin or kallikrein generation will be followed in time upon 37° C. incubation, by measuring the A405 absorbance each minute for two to three hours. Buffer will serve as a negative control, concentration series of glycated hemoglobin and/or of amyloid γ-globulins, prepared as described above, and/or 150 μg/ml kaolin will serve as positive controls.
C4. Production of Reactive Oxygen Species by Mouse Microvascular bEnd.3 Endothelial Cells
To assess production of reactive oxygen species by cultured mouse microvascular bEnd.3 endothelial cells (ECs), cells are seeded at 128,000 cells/well of a 96-well plate (Costar, 3904). After adherence for six hours, cells are washed twice with PBS and cultured overnight in DMEM with 0.1% bovine serum albumin (DMEM from Gibco with 4500 mg/l glucose, GlutaMAX and pyruvate, enriched with 100 μg/ml penicillin and streptomycin and 10% fetal calf serum). Cells are subsequently washed once with PBS enriched with 1 mM CaCl2, 0.5 mM MgCl2 and 0.1% w/v glucose (“enriched PBS”) and incubated for 30 minutes at 37° C. in the dark with 75 μl of CM-H2DCFDA (Invitrogen C6827) from a 10 μM stock in PBS. Then, cells are washed twice with enriched PBS and incubated for 15 minutes at 37° C. in the dark, either with 190 μl enriched PBS, or 190 μl enriched PBS with 1 μM Nω-Nitro-L-arginine methyl ester hydrochloride. For the analysis of ROS production, 10 μl of tester samples and controls are added to separate wells, and fluorescence is measured every two minutes for 70 minutes upon excitation at 488 nm with the emission wavelength set to 538 nm.
In one series of experiments, bEnd.3 cells were exposed to 160× diluted stocks of E. coli TOP10 or S. aureus (final cell densities 8.1×107 cells/ml and 1.13×108 cells/ml, respectively) in buffer or in buffer with either 1.25 mg/ml IgIV (Octagam), or finger domains (see below), or 220 μM Congo red, or 220 μM Thioflavin T (ThT), or 1.1 μM tPA, and ROS levels were followed in time upon 37° C. incubation. The bacteria were pre-incubated with PBS or the cross-beta structure binding compounds at concentrations of 25 mg/ml IgIV, 0.8 mg/ml finger domains, 4.4 mM Congo red, 4.4 mM Thioflavin T, or 22 μM tPA, respectively, for approximately one hour at room temperature. Subsequently, the bacterial cell suspensions were diluted twenty-fold in the cell culture medium with the ECs. As a source of finger domains, a mixture was prepared consisting of recombinant human tPA finger (F) with a C-terminal His-tag which was expressed in Saccharomyces cerevisiae (Biotechnology Application Center (BAC-Vlaardingen/Naarden, The Netherlands), a chemically synthesized hepatocyte growth factor activator (HGFA) finger domain (Dr. T. Hackeng, Academic Hospital Maastricht, the Netherlands) and recombinant human fibronectin finger domain tandem 4 and 5 with a C-terminal His-tag which was expressed in HEK 293E cells (ABC-expression facility, Utrecht). The cDNA constructs were prepared following standard procedures known to a person skilled in the art. Domain boundaries of fibronectin F4-5 and tPA F were taken from the human fibronectin and human tPA entries in the Swiss-Prot database (P02751 for fibronectin, P00750 for tPA) and comprised amino-acids NH2—I182-V276—COOH of fibronectin and NH2—G33-S85—COOH of tPA. Affinity purification of the expressed proteins was performed using His6-tag—Ni2+ interaction and a desalting step. For HGFA, residues 200 to 240 (Swiss-Prot entry Q04756) were taken. Stock solutions of fibronectin F4-5, tPA F and HGFA F were mixed to final concentrations of 0.9 mg/ml, 0.7 mg/ml and 1.25 mg/ml, respectively. The final concentration of finger domains is approximately 0.8 mg/ml.
C6. Induction of Platelet Aggregation by Pathogens
The influence of S. aureus Newman bacterium cells and E. coli TOP10 bacterium cells on blood platelet aggregation was tested with washed platelets (platelet rich plasma, PRP) in an aggregometric assay. Freshly drawn human aspirin free blood was mixed gently with citrate buffer to avoid coagulation. Blood was spinned for 15 minutes at 150*g at 20° C. and supernatant was collected; platelet rich plasma (PRP) with an adjusted final platelet number of 300,000 platelets/μl. Platelets were kept at 37° C. for at least 30 minutes, before use in the assays, to ensure that they were in the resting state. Platelets of two donors were isolated separately on different days.
For the aggregometric assays, 270 μl platelet solution was added to a glass tube and prewarmed to 37° C. A stirring magnet was added and rotation was set to 900 rpm, and the apparatus (Whole-blood aggregometer, Chrono-log, Havertown, Pa., USA) was blanked. A final volume of 30 μl was added, containing the agonist of interest (pathogen) and/or the premixed antagonist of interest (pathogen pretreated with cross-beta structure binding molecules), prediluted in HEPES-Tyrode buffer pH 7.2. Final S. aureus concentration was 1.8×109 cells/ml, for E. coli 1.3×109 cells/ml. Aggregation was followed in time by measuring the absorbance of the solution that will decrease in time upon platelet aggregation. As a positive control, 5 μM of synthetic thrombin receptor activating peptide TRAP was used. Aggregation was recorded for 15 minutes and expressed as the percentage of the transmitted light (0-100%).
Analysis of Bacterium Cell Vitality After Exposure to Cross-Beta Structure Binding Compounds
S. aureus and E. coli cells were treated with PBS or cross-beta structure binding compounds according to the above-given description. After overnight storage of the cell preparations in PBS at 4° C., vitality of the cells was assessed by inoculating 1 ml of LB medium for six hours with vigorous shaking and aeration with 25μl cell suspension. After six hours, cell density was determined by an absorbance reading at 600 nm with 50× diluted cell cultures in PBS. Starting cultures contained 3.25×108 E. coli cells/ml and 4.5×108 S. aureus cells/ml.
B. Activation of the Contact System of Coagulation by E. coli with Amyloid Curli
E. coli strain MC4100 was grown using two different conditions on agar with colonization stimulating factor (CFA), using protocols known to a person skilled in the art. E. coli on one plate were grown for approximately 44 hours at 26° C. to induce expression of amyloid curli core protein comprising cross-beta structure. A second plate was cultured for 24 hours at 37° C. which suppresses curli expression. Cells were scraped from the plates and suspended in PBS. Cell density was measured and equalized. The two E. coli preparations were tested for their ability to activate factor XII and prekallikrein in an in vitro assay for determination of contact system of coagulation-activating properties. For this purpose an E. coli density of 2.08×109 cells/ml was used in the assay, that was performed as described above.
C3. In Vitro Generation of Human Blood Derived Dendritic Cells and Activation Assay
To investigate the influence of pathogen cells comprising cross-beta structure core proteins on human DCs, the DCs were generated from non-proliferating precursors in peripheral blood mononuclear cells (PBMCs), essentially by established methods (Sallusto and Lanzavecchia, 1994). Briefly, PBMCs from buffy coat blood (Sanquin Blood Bank, Utrecht, The Netherlands) were purified using Lymphoprep (1.077 g/ml; Axis-Shield, Oslo, Norway) centrifugation. Monocytes were purified from PBMC by using Percoll (1.131 g/ml; Amersham Biosciences, Upsalla, Sweden) gradient centrifugation consisting of three layers (1.076, 1.059, and 1.045 g/ml). The low density monocyte-enriched fraction was collected, and subsequently seeded at 0.5×106/ml in CellGro DC serum-free medium (CellGenix, ITK Diagnostics, Uithoorn, The Netherlands) in polystyrene 175 cm2 culture flasks. After 45 minutes at 37° C. and 5% CO2, non-adherent cells were discarded. Adherent cells (monocytes>90% CD14 positive) were cultured in CellGro DC medium containing 10 ng/nl GM-CSF (Tebu-Bio, Heerhugowaard, The Netherlands) and 10 ng/ml IL-4 (Tebu-Bio) for six days. Fresh GM-CSF and IL-4 were added every two days. After six days at 37° C. and 5% CO2, the non-adherent cell fraction was harvested and counted. FACS analysis using the following markers was performed (FACS: anti-CD14, anti-CD1a, anti-CD80, anti-CD40, anti-CD86, anti-HLA-DR, anti-CD83, anti-CD206, anti-CD36 and anti-CD163 (all these fluorescein isothiocyanate (FITC)- and phycoerythrin (PE)- conjugated anti-CD markers were purchased from BD Biosciences, Erembodegem, Belgium), to establish whether immature DCs have been generated. Percentages of cells that were positive for the listed markers were 2% for CD14, 97% for CD1a, 4% for CD80, 69% for CD40, 69% for CD86, 60% for HLA-DR, 1% for CD83, 95% for CD206, 23% for CD36 and <1% for CD163, indicating that immature DCs have indeed been obtained. These immature DC were suspended to a final cell density of 1×106 cells/ml in CellGro DC medium, and 1 ml was transferred to low-adherent polypropylene 5-ml tubes. Maturation assays were started by adding 10 μl of a tester solution (1:100 dilution). Dilution series of E. coli TOP10 and S. aureus Newman were added to DCs and pathogen densities ranged from 5.1×104 to 3.25×106 cells/ml for the E. coli, and from 7.0×104 to 4.5×106 cells/ml for the S. aureus (two-fold dilution series in seven stages). The E. coli and S. aureus were either pre-incubated with PBS, or with a serial series of cross-beta structure binding compounds comprising ThT, Congo red and tPA+IgIV, as described above. As the negative control DCs were incubated in plain medium. DCs were stimulated for 20 hours at 37° C., 5% CO2. After this 20-hour incubation time, DCs were analyzed by FACS for the percentage of CD36, CD40 and CD206-positive cells, and for these markers the ratios between the geometric Mean Fluorescence Intensity (MFI) of a specific signal and the MFI of the accompanying noise was determined (MFI ratios). Cell morphology was assessed by analyzing forward scatter measurements and side or orthogonal scatter measurements (dead cell and contaminating lymphocyte fractions were excluded from the analysis).
Analysis of Tissue Factor Expression by THP-1 upon Stimulation with S. aureus that were Pre-Incubated with Buffer or Cross-Beta Structure Binding Compounds
For tissue factor expression analysis purposes, THP-1 cells were cultured in IMDM without gentamycin and streptomycin. At day 0, one ml of cells was seeded at 1×106 cells/ml in the wells of six-well culture plates. At day 1, cells were stimulated for six hours at 37° C. with S. aureus that were pre-incubated with PBS or with cross-beta structure binding compounds ThT, Congo red, tPA and IgIV, as described above, at a cell density of 1.8×107/ml (regular culturing conditions). Negative control was buffer. After six hours, the cells were pelleted by centrifugation and resuspended in 100 μl TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.0-7.3). Next, the cells were frozen and thawed for four subsequent cycles. Cells were centrifuged for ten minutes at 16,000*g and the supernatant was used for analysis of tissue factor (TF) expression. First, protein concentrations were determined using an established protein concentration assay (Bicinchoninic Acid (BCA) Protein Assay). Protein concentrations were equalized between samples with TBS to correct for variations in cell density. For analysis of TF levels, 50 μl of cell lysate was mixed with 50 μl TBS comprising 10 μg/ml factor X, 5 U/ml FVII, and 5 mM CaCl2, and 50 μl of a 4.5 mM stock of chromogenic activated factor X substrate S2765 in H2O, in wells of a 96-well plate. Conversion of the substrate by activated factor X at 37° C. was recorded in time for 100 minutes, by absorbance readings at 405 nm. As an additional control, factor X activity was assessed with S. aureus cells only, omitting the monocytes.
In a second series of experiments THP-1 monocytes were incubated in a similar manner with 40 μg/ml glycated hemoglobin or 10 μg/ml amyloid-β(1-40). Negative control was buffer, positive control was 10 μg/ml lipopolysaccharide.
Results
C4. In Vitro Murine bEnd.3 Endothelial Cell Activation Assay: ROS Production (I)
To determine whether cross-beta structure binding compounds Thioflavin T, Congo red, tissue-type plasminogen activator (tPA) and IgIV are able to reverse pathogenic effects of pathogens on ECs, bEnd.3 cells were exposed to 6×108 E. coli TOP10 cells/ml and ROS production by the ECs was measured in time. For this purpose, bEnd.3 cells were cultured overnight at a density of 128,000 cells/well of a 96-well plate. The overnight grown E. coli cells were either resuspended in PBS before 20× dilution in cell culture medium at 1.2×109 cells/ml (20× stock), or resuspended in 2.5 mM Congo red and 5 mM Thioflavin T in PBS after centrifugation and discarding the LB medium, incubated for ten minutes at room temperature with swirling, pelleted and dissolved at 1.2×109 cells/ml (20× stock) in 25 μM tPA and 25 mg/ml IgIV by swirling. As an example, binding of IgIV to cross-beta structure is shown for glycated albumin (
In a second series of experiments, E. coli TOP10 cells were pre-incubated in a serial set-up with PBS comprising first 2.5 mM Thioflavin T, then 5 mM Congo red and finally a mixture of 25 μM tPA and 25 mg/ml IgIV, respectively. Control cells were kept in PBS. Finally, E. coli cells were resuspended in PBS at 1.3×1010 cells/ml. Notably, pelleted cells appeared brownish-yellow and the cell suspension was orange-yellow after the incubations with cross-beta structure binding compounds, whereas the control cells in PBS were light-brown. To determine the influence of the two E. coli preparations on ROS production by bEnd.3 ECs, 8×107 E. coli cells/ml were exposed to the ECs. In
B. Factor XII/Prekallikrein Activation by E. coli
To test the potency of E. coli cells to induce factor XII/prekallikrein activation, cells at 1.3×107 cells/ml were tested in a chromogenic factor XII activation assay using chromgenic kallikrein substrate Chromozym-PK. Activation of factor XII to factor XIIa and subsequently prekallikrein to kallikrein is observed upon incubation of the proteins with E. coli cells (
C6. Induction of Platelet Aggregation by Pathogens
PRP was obtained from blood obtained from the local UMC Utrecht mini donor facility. Introduction of 1.8×109 S. aureus Newman cells/ml or 1.3×109 E. coli TOP10 cells/ml in PRP readily results in platelet aggregation (
Vitality of Bacteria Before and After Treatment with Cross-Beta Structure Binding Compounds
In
B. Binding of Cross-Beta Structure Binding Compounds to E. coli TOP10 and S. aureus Newman
Binding of cross-beta structure binding compounds to E. coli TOP10 and S. aureus Newman after incubation of the bacteria with Thioflavin T, Congo red, tPA and IgIV, as described in the Materials & Methods section, was determined in two ways. First, binding of cross-beta structure binding dyes Thioflavin T (yellow) and Congo red (red) to the bacterium cells was verified by visual inspection. The E. coli appeared as yellowish-orange cells, showing that Thioflavin T was bound to the cells and to a lesser extent Congo red. The S. aureus cells were intense red, indicative for Congo red binding. Due to the intense red color, it is possible that yellow Thioflavin T can not be seen. In conclusion, S. aureus binds more Congo red than E. coli, whereas no comparative qualitative measure can be given for Thioflavin T binding. Obviously, Thioflavin T is bound to E. coli.
Whether tPA is bound to the E. coli and S. aureus after incubation with 25 μM tPA, was assessed with a tPA/plasminogen chromogenic activation assay, as described above. Plasminogen and chromogenic plasmin substrate Biopep-1751 were mixed with E. coli or S. aureus incubated with buffer only, or with E. coli or S. aureus that were pre-incubated with, amongst other cross-beta structure binding compounds, tPA. Plasmin generation by tPA, measured as conversion of the substrate, can only occur when an external source of tPA activity is introduced in the reaction mixture (
B. Activation of the Contact System of Blood Coagulation by E. coli with Amyloid Curli Protein
It has been established that E. coli bacteria express an amyloid core protein, curli, at the cell surface, depending on culturing conditions. When E. coli MC4100 is cultured on CFA agar for 44 hours at 26° C., expression of curli is facilitated, whereas no curli is expressed when cells are grown for 24 hours at 37° C. Curli with cross-beta structure has been defined as an important determinant for binding properties of the E. coli towards fibronectin of the host, a cross-beta structure binding protein through the ability of the finger domains (fibronectin type I domains 4, 5, 10, 11 and 12 to bind to proteins comprising cross-beta structure. The E. coli with and without amyloid curli comprising cross-beta structure were applied to a chromogenic factor XII/prekallikrein activation assay. When factor XII becomes activated kallikrein is formed from prekallikrein by activated factor XII, and kallikrein substrate Chromozym-PK is converted, which is measured by absorbance readings in time. In
C4. In Vitro Murine bEnd.3 Endothelial Cell Activation Assay: ROS Production (II)
To test whether cross-beta structure binding compounds ThT, Congo red, IgIV, tPA and finger domains of fibronectin, HGFA and tPA have the potency to reverse adverse effects of pathogens E. coli TOP10 and S. aureus Newman on bEnd.3 ECs with respect to ROS expression, the ECs were exposed to 8.1×107 E. coli cells/ml or 1.13×108 S. aureus cells/ml in the presence of buffer, or in the presence of either 1.25 mg/ml IgIV, or 0.8 mg/ml finger domains, or 220 μM Congo red, or 220 μM ThT, or 1.1 μM tPA, and ROS levels were followed in time upon 37° C. incubation. The bacteria were also pre-incubated with ThT, Congo red, IgIV and tPA as described above. From
C7. Ex Vivo Human Plasma Coagulation Assays
For analysis of the influence of cross-beta structure comprising pathogens on the characteristics of blood coagulation, and for analysis of the effects of cross-beta structure binding compounds on the influence of pathogens on coagulation, aPTT and PT coagulation tests were performed. Pooled human plasma of approximately 40 apparently healthy donors was clotted by adding either negatively charged phospholipids, CaCl2 and kaolin in the aPTT set-up, or tissue factor rich thromboplastin and CaCl2 in the PT set-up. Before coagulation tests were performed, two-fold diluted plasma was pre-incubated for approximately one hour at room temperature with PBS (control), 6.5×109 E. coli TOP10 cells/ml, or 6.5×109 E. coli TOP10 cells/ml that were pre-incubated with cross-beta structure binding compounds Congo red, ThT, tPA and IgIV. Before coagulation tests were performed, bacterium cells were pelleted by centrifugation and plasma supernatants were analyzed in the aPTT and PT assays. Results are shown in
From the delayed clotting time in an aPTT with PBS-pre-incubated E. coli control cells we conclude that factors that are essential for the contact system of coagulation are bound to E. coli and subsequently partly depleted from plasma upon pelleting the bacteria. Likely candidates are cross-beta structure binding proteins factor XII and high molecular weight kininogen (see Table 4), and perhaps fibronectin and fibrin. Acceleration of the coagulation time, as seen in PT set-ups after incubation of plasma with E. coli, point to a pro-coagulant activity of the bacterium. Either, cross-beta structure comprising proteins are secreted from the E. coli into the medium, or other pro-coagulant molecules are secreted, or anticoagulant molecules are depleted from plasma upon pelleting the E. coli cells, or (pro)fibrinolytic molecules, like, for example, tPA and plasmin(ogen) are depleted from plasma upon pelleting the E. coli cells, or fibrinolysis inhibitors are secreted into plasma by the pathogen, like, for example, bacterial plasminogen activator inhibitor analogues and/or α2-anti-plasmin analogues, or the plasma is already in a pro-coagulant state upon incubation with E. coli, due to the recruitment of cross-beta structure comprising proteins involved in the contact system of coagulation, i.e., factor XII and HMWK (see Table 4), by cross-beta structure at the E. coli, resulting in activation of the coagulation system. When E. coli are pre-treated with cross-beta structure binding compounds, coagulation is strongly delayed in both PT and aPTT analyses (
C3. Maturation of Human Dendritic Cells Induced by Pathogens is Influenced by Cross-Beta Structure Binding Compounds
Immature human DCs were obtained from PBMCs following established protocols. Upon stimulation of the immature DCs for 20 hours with a concentration series of pathogens E. coli TOP10 or S. aureus Newman, which comprise surface cross-beta structure proteins, maturation markers were analyzed with FACS, i.e., down-regulation of endocytic receptors CD36 and CD206 and up regulation of co-stimulatory receptor molecule CD40. The cell surface expression of these markers on control cells that were incubated for 20 hours with medium only, served as a reference for immature DCs. DCs were exposed to 5.1×104−3.25×106 E. coli cells/ml and 7.0×104−4.5×106 S. aureus cells/ml (two-fold dilution series in seven stages). The E. coli and S. aureus were either pre-incubated with PBS, or with cross-beta structure binding compounds ThT, Congo red, tPA and IgIV. At higher pathogen densities, e.g., 1.3×107 E. coli cells/ml or 1.8×107 S. aureus cells/ml, a three- to four-fold increase in FITC background fluorescence was observed when assessing those pathogen samples that were pre-incubated with cross-beta structure binding molecules. Perhaps, ThT excitation and emission wavelengths (435 and 485 nm) are to close to those of FITC (NB FACS contains an argon-ion laser, i.e., 488 nm excitation wavelength; FITC emission channel 530/30 nm, and PE emission channel 675/25 nm), or the signals can be due to auto-fluorescence of bacterium cells. Therefore, the increased background fluorescence is indicative for an interaction of pathogen cells with the DCs. Whether bacterium cells are bound to the DC surface and/or internalized and/or degraded remains to be established. At lower bacterium cell densities, background fluorescence in the FITC and phycoerythrin (PE) channels is not increased in samples comprising pathogens in comparison to non-treated DCs.
Co-stimulatory receptor CD40 is up-regulated on DCs upon stimulation with all four pathogen preparations, i.e., untreated E. coli, E. coli pre-incubated with cross-beta structure binding compounds, untreated S. aureus and S. aureus pre-incubated with cross-beta structure markers, indicative for maturation of the DCs. The percentage of CD40-positive cells is increased from approximately 60% for the control cells (medium control) that were not stimulated to 70-90% upon stimulation with pathogens (with or without cross-beta structure binding compounds; data not shown). Cell surface expression (MFI ratios) of CD40 increased in a pathogen concentration dependent manner for both E. coli and S. aureus (
Endocytic receptor CD36 is down-regulated on DCs upon stimulation with all four pathogen preparations, i.e., untreated E. coli, E. coli pre-incubated with cross-beta structure binding compounds, untreated S. aureus and S. aureus pre-incubated with cross-beta structure markers, indicative for maturation of the DCs due to exposure to the pathogens. The percentage of CD36-positive cells decreases from approximately 50% with the lowest pathogen cell density, which is comparative to the percentage of CD36-positive cells found for the control DCs (medium control) that were not stimulated, to 5 to 10% upon stimulation with the highest pathogen densities (data not shown). Pre-incubation of pathogens with cross-beta structure binding compounds results in less down-regulation of the number of CD36-positive DCs, compared to buffer-treated pathogen cells, indicative for less potent maturation of the DCs by pathogens that were pre-incubated with cross-beta structure binding compounds ThT, Congo red, tPA and IgIV (data not shown). Cell surface expression (MFI ratios) of CD36 decreased in a pathogen concentration dependent manner for both E. coli and S. aureus (
The percentage of endocytic receptor CD206-positive DCs is hardly influenced upon stimulation with all four pathogen preparations, i.e., untreated E. coli, E. coli pre-incubated with cross-beta structure binding compounds, untreated S. aureus and S. aureus pre-incubated with cross-beta structure markers, indicating that the mannose receptor does not play an important role in maturation of DCs upon exposure to the pathogens with cross-beta structure surface proteins (data not shown). The percentage of CD206-positive cells stays high at approximately 75 to 90%, whereas non-stimulated cells (medium control) were approximately 90% CD206 positive (data not shown). It seems that lowering the pathogen-induced CD206 cell surface expression results in somewhat decreased number of CD206-positive cells. For all four pathogen preparations endocytic receptor CD206 surface density (MFI ratios) is decreased to a similar extent, indicative for maturation of the DCs upon stimulation with the pathogens (
Analysis of Tissue Factor Expression by THP-1 upon Stimulation with S. aureus that were Pre-Incubated with Buffer or Cross-Beta Structure Binding Compounds
For tissue factor expression analysis purposes, THP-1 cells were exposed to 1.8×107 S. aureus cells/ml for six hours at 37° C. TF activity was determined in an indirect way by assessing activation of factor X in the presence of activated factor VII, with three-fold diluted THP-1 monocyte cell lysate. Factor X activity in THP-1 cell lysates after exposure to PBS-incubated S. aureus was higher than in lysates of cells that were exposed to S. aureus which was pre-incubated with cross-beta structure compounds Thioflavin T, Congo red, tPA and IgIV (
Misfolded proteins with cross-beta structure conformation, i.e., glycated hemoglobin (Hb-advanced glycation end-product, AGE) and amyloid-β induce elevated levels of TF, as determined by potent factor X activation by THP-1 monocyte lysates after incubation of the cells with the misfolded proteins (
In Table 7 and
Abbreviations
APC, antigen-presenting cell; aPTT, activated partial thromboplastin time; BCA, Bicinchoninic Acid; BiP/grp78, Immunoglobulin heavy chain-binding protein/Endoplasmic reticulum lumenal Ca(2+)-binding protein; cbs, cross-beta structure; CD, Cluster of Differentiation; CFA, colonization stimulating factor; DC, dendritic cell; DMEM, Dulbecco's Modified Eagle Medium; EC, endothelial cell; E. coli, Escherichia coli; ELISA, enzyme-linked immunosorbent assay; F, finger domain/fibronectin type I domain; FVII, factor VII; FACS, Fluorescence Activated Cell Sorting; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; Fn, fibronectin; GM-CSF, GRANULOCYTE MACROPHAGE COLONY STIMULATING FACTOR; HBS, HEPES-buffered saline; HEPES, {2-(4-(2-Hydroxyethyl)-1-piperazinyl) ethanesulfonic Acid}; HGFA, hepatocyte growth factor activator; HMWK, high molecular weight kininogen; HSP, heat-shock protein; HLA-DR, D-related human leukocyte antigen; HUVEC, human umbilical vein endothelial cell; IgIV, immunoglobulins intravenous; IMDM, Iscove's Modified Dulbecco's Medium; IL, interleukin; IvIG, intravenous immunoglobulins; IFN, interferon; LB, luria broth; LRP/CD91, low density lipoprotein related receptor; LOX, lecton-like receptor for oxidized low density lipoprotein; MHC, human leukocyte antigen; MTT, mitochondrial metabolic activity; MFI, mean fluorescent intensity; NO, nitric oxide; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PE, phycoerythrin; PRP, platelet rich plasma; PT, prothrombin time; PMA, phorbol 12-myristate 13-acetate; RPMI, Roswell Park Memorial Institute; ROS, reactive oxygen species; S. aureus, Staphylococcus aureus; S. pyogenes, Streptococcus pyogenes; sRAGE, soluble fragment of receptor for advanced glycation end products; TBS, Tris(hydroxymethyl)aminomethane Hydrochloride-buffered saline; ThT, thioflavin T; TLR, Toll-like receptor; TNF-α, tumor necrosis factor-α; TRAP, synthetic thrombin receptor activating peptide; TPA, Tetra-Phorbol-Acetate; tPA, tissue-type plasminogen activator; ULS, universal linkage system.
Escherichia coli
Salmonella enterica
Typhimurium)
Staphylococcus
aureus
Streptococcus
pyogenes
‡Monoclonal antibodies developed in collaboration with the ABC-Hybridoma Facility, Utrecht University, Utrecht, The Netherlands.
a)Antigen albumin-AGE and ligand Aβ were send in to Davids Biotechnologie (Regensburg, Germany); a rabbit was immunized with albumin-AGE, antibodies against a structural epitope were affinity purified using a column with immobilized Aβ.
2)PPACK is Phe-Pro-Arg-chloromethylketone (SEQ-ID 8), εACA is ε-amino caproic acid, tPA is tissue-type plasminogen activator
‡Monoclonal antibodies developed in collaboration with the ABC-Hybridoma Facility, Utrecht University, Utrecht, The Netherlands.
Borellia burgdorferi
Candida albicans
Escherichia coli
Hemophilus influenza
Helicobacter pylori
Mycobacterium avium
M.. tuberculosis
Neisseria meningitidus
N. gonorrhoeae
Pneumocystis carinii
Proteus mirabilis
Pseudomonas aeruginosa
Salmonella enterica (serovar
Typhimurium)
Staphylococcus aureus
Streptococcus pneumonia
Streptococcus pyogenes
Yersinia pestis
Y. enterocolitica
References:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=pubmed and Madigan M T, Martinko J M, Parker J, Brock Biology of Microorganisms, Prentice Hall, 10th edition, ISBN 0-13-049147-0.
E. coli
E. coli
E. coli
S. aureus +
E. coli
S. aureus
‡Vitality of the bacteria was determined after treatment with buffer or with cross-beta structure binding compounds.
1)“cbs binders” refers to pre-incubation of pathogen with cross-beta structure binding compounds, before applying the pathogen in the assays.
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| Number | Date | Country | Kind |
|---|---|---|---|
| 05075656.8 | Mar 2005 | EP | regional |
This application claims priority to U.S. Provisional Patent Application Ser. No. 60/663,487, filed Mar. 18, 2005, and European Patent Application Serial No. EP05075656.8, also filed Mar. 18, 2005, the contents of the entirety of each of which are incorporated by this reference.
| Number | Date | Country | |
|---|---|---|---|
| 60663487 | Mar 2005 | US |
