DE102006012918A1 - Test system, useful for the determination of the effectiveness of biologic and systemic therapies in psoriasis, comprises cells that express the human Thy-1 (CD90) - Google Patents

Abstract

Test system comprises at least one cell that expresses the human Thy-1 (CD90). Independent claims are included for: (1) a procedure for the determination of the effectiveness of biologics and systemic therapies in psoriasis comprising: determining cell-to-cell interaction of chinese hamster ovary (CHO) cells, which are transfected with human Thy-1 (CD90), with the granulocytes of the patient with biological or systemic therapy; determining the cell-to-cell interaction of CHO cells, which are transfected with human Thy-1 (CD90), with control cells; comparing the cell-to-cell interactions, where a decreased cell-to-cell interaction of CHO cells is an indication for the anti-inflammatory effect of the biologic or the systemic therapy and a non-decreased cell-to-cell interaction of CHO cells, is an indication for the therapy failure of the biologic or the systemic therapy; and (2) a procedure for the identification of substances with anti-inflammatory or anti-metastatic effect comprising: determining the cell-to-cell interaction of cells that express human Thy-1 (CD90) at their surface, with cells that express a Thy-1 (CD90)-ligand at the surface, in the presence and absence of the test substance; and comparing the cell-to-cell interactions, where the cell-to-cell interaction is decreased in presence of the test substance, which is an indication for the anti-inflammatory or anti- metastatic effect of the test substance.

Description

The The invention relates to a test system and method for determining the Effectiveness of biologics and systemic therapies in psoriasis therapy as well as for the identification of substances with anti-inflammatory or anti-metastatic effect for use in medicine.

The Immigration of circulating cells, such as inflammatory cells and immune cells, from vessels to certain Fabric is for Immune and inflammatory reactions and metastasis of central importance. A key role plays during this Process the adhesion of the circulating cells to the vascular endothelial cells (EC) via specific Docking structures (cell adhesion molecules). Therefore is the detailed study of the molecules and mechanisms involved for the understanding and for estimation of possibilities for the regulation of these physiological and pathological processes of great Interest.

in the healthy tissue, the endothelium is in a dormant, non-activated Condition that hardly emanates from inflammatory cells in the surrounding area Tissue allows. In the course of an injury or infection comes with subsequent inflammation it on the other hand to activate the EC. This activation causes that the peripheral leukocytes from the circulation to the place of inflammation guided as it is observed in acute and chronic inflammation. About the use different combinations of cell adhesion molecules on both the EC and on the circulating cells themselves and through a wide range Of activating factors is the accumulation of immune and inflammatory cells Determined and regulated in the tissue. In pathological situations as with allergies, chronic inflammatory diseases (such as Psoriasis, asthma, rheumatoid arthritis) are these processes disturbed, and there is a massive and unphysiological immigration of these Cells into the tissue.

In similar Way, tumor cells use the repertoire of adhesion molecules to help in the course of metastasis from the lymph or blood vessel system in secondary Enter organs and form metastases there.

Therefore So it's a big one Interest in finding resources that interfere with this system and the Emigration of these cells can inhibit and so a therapy of "pathological Situations ".

psoriasis (Psoriasis) is a chronic inflammatory disease of the skin. Malregulated T cells are at the onset of disease and disease progression involved. Accordingly, the research interest is focused in the development of new therapeutics for T-cell dysregulation.

When Standard therapy will be topical in the treatment of psoriasis preparations used, i. The skin is treated locally with ointments or solutions. As topical agents, vitamin D3 analogs such as Calcitriol and calcipotriol, steroids and retinoids used. The respective active ingredients do not penetrate very deeply into the skin which limits their effectiveness. They must be applied regularly and are not suitable for the treatment of extensive psoriatic disease.

to Treatment of psoriasis symptoms will also light therapy (UVB therapy) applied. Light therapies can but only about be applied a certain amount of time since the risk of sunburn, premature skin aging and the development of skin cancer. Around five Percent of psoriasis patients also have a photosensitive Psoriasis up.

Especially heavier forms of psoriasis can with systemic therapies as well as with biologics (biotechnological manufactured medicines). As systemic therapeutics For example, oral retinoids such as acitretin, cyclosporin, Methotrexate and fumaric acid esters used.

biologics are usually antibodies or fusion proteins involved in the regulatory processes of T cells intervene and thus inhibit the activity of T cells or their activity as an inflammatory mediator neutralize. For example, the biologic Raptiva (efalizumab) is a humanized antibody, the purposefully an early one Blocking T-cell activation including its multiplication causes and the emigration of T cells from the blood vessels in the Prevents psoriasis.

These However, medicines are very expensive. A recognizable improvement psoriasis occurs only after prolonged therapy. That only after A (costly) treatment over a few weeks can with Safety be determined whether the therapy ever strikes or whether a therapy failure occurs.

The object of the invention is therefore to provide a method and a test system which make it possible to test pharmaceuticals for potential anti-inflammatory and / or anti-metastatic action. In particular, it is the object of the invention to provide a method and a test system with which early after the onset of psoriasis treatment with systemic therapy or with biologics It can be established whether the treatment is likely to lead to an improvement of the clinical picture or whether a treatment failure is to be expected.

According to the invention Task solved by a test system containing at least cells containing human Thy-1 (CD90) express.

humane Thy-1 (CD90) expressing cells are preferably activated microvascular dermal endothelial cells, human dermal fibroblasts or stably transfected with Thy-1 CHO cell line. Activated microvascular dermal endothelial cells are available by in vitro stimulation of microvascular dermal endothelial cells with Phorbol 12,13-myristoilacetat or tumor necrosis factor-α in concentrations known to those skilled in the art.

Especially preferred is a stably transfected with Thy-1 CHO cell line, the available is by cloning the cDNA for Human Thy-1 (CD90) (Accession number: M11749) from nucleotide 27 to nucleotide 1519 in the expression vector pcDNA6 (from Invitrogen Carlsbad, USA), transfecting CHO-K1 cells (ATCC CCL61) using Electroporation and and selection of a stably expressing cell line using blasticidin (Invitrogen).

The Expression of human Thy-1 (CD90) is by flow cytometry with an anti-CD90 (anti-Thy-1) Antibody verifiable.

Preferably contains the test system in addition Control cells expressing human Thy-1 (CD90) cells but do not contain human Thy-1. Is it? for example, in the cells expressing human Thy-1 (CD90), a stably transfected with Thy-1 CHO cell line, which is available by cloning the cDNA for Human Thy-1 (CD90) (Accession number: M11749) from nucleotide 27 to nucleotide 1519 in the expression vector pcDNA6 (from Invitrogen Carlsbad, USA), transfecting CHO-K1 cells (ATCC CCL61) using Electroporation and and selection of a stably expressing cell line using blasticidin (Invitrogen), so be as control cells preferably one with only the empty expression vector pcDNA6 transfected CHO cell line used.

By the control cells is advantageously a standardization of the test system possible.

In a further advantageous embodiment of the invention the Thy-1 expressing Cells, in particular the Thy-1 transfected CHO cell line, or the control cells, in particular the vector-transfected CHO cells, as a monolayer on a solid support, for example cell culture vessels, Chamber slides, etc. fixed. Preferably, it is fixed with ice-cold methanol. This embodiment can easily be produced in advance and stored at -20 ° C. without the test results available with the test system to influence.

In Another embodiment of the invention are the Thy-1-expressing Cells, in particular the Thy-1 transfected CHO cell line, or the Control cells, in particular the vector-transfected CHO cells, Sown in special tissue culture plates, which is an automated Allow analysis, for example by means of a fluorimeter on the fluorescence of fluorescently labeled cells bound to the Thy-1 expressing cells Cells or other familiar to the expert evaluation.

• a. Bestimmen der Zell-Zell-Interaktion von CHO-Zellen, die mit humanem Thy-1 (CD90) transfiziert sind, mit Granulozyten von Patienten vor Therapiebeginn und im Verlauf der Therapie mit einem einem Biologikum oder einer systemischen Therapie
• b. Bestimmen der Zell-Zell-Interaktion von CHO-Zellen, die mit humanem Thy-1 (CD90) transfiziert sind, mit Kontrollzellen,
• c. Vergleichen der Zell-Zell-Interaktionen, wobei – eine verminderte Zell-Zell-Interaktion von CHO-Zellen, die mit humanem Thy-1 (CD90) transfiziert sind, mit Granulozyten von Patienten, die mit einem Biologikum oder systemischer Therapie behandelt werden, ein Indiz für die anti-entzündliche Wirkung des Biologikum oder der systemischen Therapie ist und – eine nicht verminderte Zell-Zell-Interaktion von CHO-Zellen, die mit humanem Thy-1 (CD90) transfiziert sind, mit Granulozyten von Patienten, die mit einem Biologikum oder systemischer Therapie behandelt werden, ein Indiz für Therapieverasagen des Biologikum oder der systemischen Therapie ist.

The invention furthermore relates to the use of the test system according to the invention for determining the effectiveness of biologics and systemic therapies in psoriasis therapy and to a method for determining the effectiveness of biologics and systemic therapies in psoriasis therapy, comprising the following steps:

• a. Determining the cell-cell interaction of CHO cells transfected with human Thy-1 (CD90) with granulocytes from patients before initiation of therapy and during therapy with a biological or systemic therapy
• b. Determining the cell-cell interaction of CHO cells transfected with human Thy-1 (CD90) with control cells,
• c. Comparing cell-cell interactions, wherein: decreased cell-cell interaction of CHO cells transfected with human Thy-1 (CD90) with granulocytes from patients treated with a biological or systemic therapy Indication of the anti-inflammatory effect of the biological or systemic therapy is and - an undiminished cell-cell interaction of CHO cells transfected with human Thy-1 (CD90) with granulocytes from patients treated with a biological agent or systemic therapy is an indication for therapy orgasmic or systemic therapy.

In a preferred embodiment the method according to the invention to determine the efficacy of biologics and systemic therapies Psoriasis therapy will be used to determine the cell-cell interaction the granulocytes are labeled according to methods known to those skilled in the art, Preferably, the granulocytes are biotinylated. A biotinylation can be carried out for example by means of D-biotinoyl-ε-aminocaponsäurehydroxysuccinimidester.

To Addition of the labeled granulocytes and incubation become unbound Cells removed by means of suitable washing steps. Preferably for the determination of cell-cell interaction adherent biotinylated granulocytes via their mark demonstrated, for example, they are by a Stained with streptavidin-coupled fluorescent dye and subsequently counted in the fluorescence microscope.

The Determining the cell-cell interaction is preferably carried out by means of automated analysis.

In a particularly preferred embodiment of the method, a CHO cell line stably transfected with human Thy-1 (CD90) and, as control cells, a CHO cell line stably transfected with the vector (eg, pcDNA6) without the Thy-1 insert are placed on chamber seeded (NUNC, 8 well, Permanox) and cultured for 24 h. The formation of a completely dense cell lawn is controlled microscopically. The granulocytes are prepared by methods known to the person skilled in the art and labeled, for example, with D-biotinoyl-ε-aminocaproic acid hydroxysuccinimide ester. To each well of CHO-Thy-1 cells is added 5x10 ⁵ labeled cells. After incubation for 30 minutes at 37 ° C, the unbound cells are removed with several washes (PBS, 37 ° C). The adhered, biotin-labeled cells are incubated with CY3T "'- conjugated streptavidin (Beckman-Couiter, 1: 500 in PBS / 1% BSA) for 30 min at room temperature and then washed several times with PBS: The adherent cells are then detected by fluorescence microscopy and are counted in 10 microscopic fields. To represent the Thy-1 specific adhesion, the adhesion to the vector-transfected control cells is considered blank and subtracted from the specific adhesion.

With the test system according to the invention and the associated Advantageously, an easily determinable in vitro laboratory parameter is provided for testing procedures. the already shortly after the beginning of a psoriasis therapy with systemic Therapy or biologics provides statements on the effectiveness of the therapies. This way is an early one Forecast over the therapeutic success possible and it does not have to wait several weeks until one outwardly visible improvement of the psoriatic condition occurs or not to be able to assess the effectiveness of the therapy. Decreases the adhesion of the Patient granulocytes to Thy-1 transfected CHO cells during the Treatment, this is an indication of the onset of therapy. Decreases the adhesion does not decrease, this indicates a treatment failure. The therapy can then be changed.

According to the prior art it is assumed that psoriasis is mainly caused by a malfunction of the T cells. Accordingly, the therapeutic approaches also focus on influencing the T-cell-mediated immune reactions (eg cyclosporin A, Raptiva ^® ).

The However, the test system shown here is based on the measurement of neutrophil activity Granulocytes, which are reflected in their adhesive properties. Thy-1-mediated adhesion but plays no role in the adhesion of T cells, but is at the adhesion involved in neutrophil granulocytes and monocytes.

All the more surprising is it for the person skilled in the art that the test system according to the invention is capable of over the Determination of Granulocyte Function Therapy effects that are primarily based on the T cells are directed to display. Apparently influences the influence the T-cell mediated Immune reactions by the therapeutics therefore also directly or indirectly the function of neutrophilic granulocytes.

In a further preferred embodiment of the invention the test system according to the invention additionally Cells expressing a Thy-1 (CD90) ligand on the surface.

In an advantageous embodiment are cells that express a Thy-1 (CD90) ligand on the surface, Monocytes, neutrophil granulocytes or melanoma cells. In a preferred embodiment are cells expressing a surface Thy-1 (CD90) ligand with a Thy-1 ligand, for example Mac1 (CD11b / CD18) or aVb3 (CD51 / CD61), transfected CHO cell line. These are available through Transfecting CHO cells with an expression vector containing the cDNA of the Thy-1 ligand contains.

• a. Bestimmen der Zell-Zell-Interaktion von Zellen, welche humanes Thy-1 (CD90) an ihrer Oberfläche exprimieren, mit Zellen, die einen Thy-1 (CD90)-Liganden an der Oberfläche exprimieren, in Abwesenheit der Testsubstanz,
• b. Bestimmen der Zell-Zell-Interaktion von Zellen, welche humanes Thy-1 (CD90) an ihrer Oberfläche exprimieren, mit Zellen, die einen Thy-1 (CD90)-Liganden an der Oberfläche exprimieren, in Anwesenheit der Testsubstanz,
• c. Vergleichen der Zell-Zell-Interaktionen,

wobei eine in Anwesenheit der Testsubstanz verminderte Zell-Zell-Interaktion ein Indiz für die anti-entzündliche oder anti-metastatische Wirkung der Testsubstanz ist.The invention furthermore relates to the use of the test system according to the invention for the identification of substances having an antiinflammatory or anti-metastatic action and to a method for the identification of substances having an antiinflammatory or anti-metastatic action with the steps

• a. Determining the cell-cell interaction of cells expressing human Thy-1 (CD90) on their surface with cells expressing a Thy-1 (CD90) ligand on the surface, in the absence of the test substance,
• b. Determining the cell-cell interaction of cells expressing human Thy-1 (CD90) on their surface with cells expressing a Thy-1 (CD90) ligand on the surface in the presence of the test substance,
• c. Comparing cell-cell interactions,

wherein one in the presence of the test substance vermin cell-cell interaction is an indication of the anti-inflammatory or anti-metastatic effect of the test substance.

When Cells expressing a Thy-1 (CD90) ligand at the surface be used to identify substances with anti-inflammatory Effect preferably cells used, which are the Thy-1 ligand Mac1 (CD11b / CD18), like monocytes, express neutrophilic granulocytes etc. For the identification of substances with anti-metastatic effect preferably cells which contain the Thy-1 ligand aVb3 (CD51 / CD61) express like melanoma cells.

In a preferred embodiment of the method are one with a Thy-1 ligand, for example Mac1 (CD11b / CD18) or aVb3 (CD51 / CD61), transfected CHO cell line as cells expressing a Thy-1 Express (CD90) ligands on the surface used. For the establishment of cells transfected with Thy-1 ligands become CHO cells with an expression vector containing the cDNA of the Thy-1 ligand transfected.

The Test substance is preferably first either with the Thy-1 expressing Cells or cells incubated with the Thy-1 ligand-expressing cells, before the process step b) is performed. The incubation period is preferably in the short-term range, for example 30 to 60 minutes, changes caused by the test substance to be able to determine those on influencing binding properties such as affinity or cytoskeletal changes based. Is about a longer Period, for example 24 hours, incubated, can also beneficial effects on a new synthesis of proteins be based, be determined.

To the Determining the cell-cell interaction of cells expressing human Thy-1 (CD90) on its surface and cells expressing a Thy-1 (CD90) ligand their surface in a preferred embodiment of the method, the Cells that express a Thy-1 (CD90) ligand on their surface labeled methods known to those skilled in the art, e.g. with biotin. A Biotinylation can be carried out, for example, by means of D-biotinoyl-ε-aminocaponic acid hydroxysuccinimide ester carried out become. After addition of the Thy-1 ligand-expressing cells and incubation Unbound cells are removed by means of suitable washing steps. adherent Cells are over their label detected, e.g. by binding a streptavidin-coupled one Fluorescent dye, and e.g. by counting in the fluorescence microscope certainly.

Preferably determining the cell-cell interaction by means of automated Analysis.

In a particularly preferred embodiment of the method, a CHO cell line stably transfected with human Thy-1 (CD90) and, as control cells, a CHO cell line stably transfected with the vector (eg, pcDNA6) without the Thy-1 insert are placed on chamber seeded (NUNC, 8 well, Permanox) and cultured for 24 h. The formation of a completely dense cell lawn is controlled microscopically. The CD90 ligand-expressing cells are prepared according to their origin and labeled with D-biotinoyl-ε-aminocapronsäurehydroxysuccinimidester (Roche Biochemicals, Mannheim) according to the manufacturer's instructions. To each well of CHO-Thy-1 cells is added 5x10 ⁵ labeled cells. After incubation for 30 minutes at 37 ° C, the unbound cells are removed with several washes (PBS, 37 ° C). The adhered, biotin-labeled cells are incubated with CY3T "'- conjugated streptavidin (Beckman-Couiter, 1: 500 in PBS / 1% BSA) for 30 min at room temperature and then washed several times with PBS: The adherent cells are then detected by fluorescence microscopy and are counted in 10 microscopic fields. To represent the Thy-1 specific adhesion, the adhesion to the vector-transfected control cells is considered blank and subtracted from the specific adhesion. For stimulation or blocking approaches, the respective biotinylated cells are treated with antibodies or cytokines prior to the experiment.

With the method according to the invention it is advantageously possible a wide range of substances to test their ability to adhesion to inhibit the Thy-1. Positively tested substances can be used in the Connection, for example, will be checked to see if they are also in the Location are, the adhesion of blood cells on endothelial cells - the physiologically relevant Cells in adhesion processes in vivo - too inhibit. As the recovery and culture of microvascular endothelial cells is very complicated and expensive, in particular the Thy-1 transfected Cells are excellent model systems to a large number to screen substances in high throughput and on those in the respective Narrow the context of interest substances. With this restricted Set of substances are more tests in complicated systems like on endothelial cells and later possible in animal models.

The invention is based on the scientific finding that human Thy-1 (CD90) is functionally relevant for cell adhesion processes in inflammatory and metastatic processes. In healthy human skin, Thy-1 is expressed on fibroblasts and nerve cells, but not on EC. In contrast, it comes to massi Re-formation of Thy-1 on the EC in tissues in which cell activation, inflammation or neoangiogenesis is observed.

The Leukocyte integrin Mac-1 (amb2; CD11b / CD18) on neutrophilic granulocytes and monocytes became a specific interaction partner of Thy-1 identified. The investigation of the functional consequence of this Interaction has revealed that the Thy-1 on activated microvascular EC the adhesion of neutrophilic granulocytes and monocytes via interaction with the Mac-1 (CD11b / CD18) mediates. This could prove that the human Thy-1 is an important receptor for the Mac-1 on activated EC is crucial for the recruitment of leukocytes into inflammatory areas can contribute. In a similar way was the avb3 integrin as an interaction partner of the Thy-1 on tumor cells, specifically detected on melanoma cells.

These Interaction mediates the adhesion From tumor cells to the endothelium and thus can be crucial to the Process of tumor metastasis may be involved.

To the Proof of this functional importance of Thy-1 has been genetic changed Cells expressing Thy-1 on their cell surface (Thy-1-transfected CHO cells) and corresponding control cells (vector-transfected CHO cells) generated. Neutrophil granulocytes and monocytes bind clearly stronger to the Thy-1 transfected cells than to the control cells, the no Thy-1 on their cell surface mint.

Around to support the physiological relevance of this experimental approach, became the meaning of Thy-1 for the adhesion of neutrophilic granulocytes and monocytes and melanoma cells microvascular Endothelial cells, the physiologically relevant target cells in vivo, repeated in our experimental setup. Also with the endothelial cells could be shown that the Thy-1 plays an important role in the adhesion of neutrophilic granulocytes and monocytes and melanoma cells to the Endothelium has. So leads et al blockade of Thy-1 on the endothelial cells to a drastic reduction (up to 60%) of the adhesion of neutrophilic granulocytes and monocytes and melanoma cells.

at This experimental approach must take into account that microvascular endothelial cells express a wide range of different molecules on their cell surface, the are capable of adhering to inflammatory cells or tumor cells to convey. The drastic reduction of adhesion after blockade of a single molecule, of the Thy-1, on the endothelial cells points to the enormous physiological Meaning of Thy-1 in this process. Since the adhesion of neutrophilic granulocytes and monocytes in vivo are the prerequisite for the migration of these inflammatory cells Tissue is as it is observed in chronic inflammatory diseases it is of therapeutic importance to inhibit this process.

With the method according to the invention and test system for the identification of substances with anti-inflammatory or anti-metastatic action may therefore be an important adhesion mechanism presented in vitro and factors can be investigated which inhibit this mechanism.

in the Test system is e.g. the adhesion of neutrophilic granulocytes to the Thy-1 transfected cells by addition of known anti-inflammatory Substances clearly inhibited. So the test system is for example capable of anti-inflammatory Effect of cyclosporin A, a known immunomodulatory and anti-inflammatory acting drug, to prove. The physiological relevance This finding was again confirmed by equivalent experiments on microvascular endothelial cells demonstrated.

Farther allows the test system according to the invention Factors to test the interaction of endothelial and circulating Cells can block and thus potentially a therapeutic anti-inflammatory or anti-adhesive Have effect. These substances are of great interest to the unwanted, uncontrolled and unphysiological emigration of inflammatory cells chronic diseases, allergies, autoimmune diseases or Transplant rejection to prevent and thus ultimately the inflammatory reactions in the tissue to minimize.

Based the following figures and embodiments the invention will be closer explained, without to restrict this.

there demonstrate

1 : Neutrophil granulocytes from patients with psoriasis adhere significantly more to Thy-1 than healthy neutrophil granulocytes

Neutrophil granulocytes (PMNC) from psoriatic and healthy donors were isolated, labeled with biotin, and added to Thy-1, ICAM-1, and vector-transfected control cells for approximately 60 minutes at 37 ° C. After incubation, unbound cells were gently removed by washing, adherent cells were fixed and counted. To illustrate the Thy-1 or ICAM-1-specific adhesion, the number of adherent to the vector-transfected cells control cells of the Subtracted from Thy-1 and ICAM-1 adherent cells. The number of adherent healthy PMNC was set at 100% and the change in psoriatic PMNC adhesion compared to healthy PMNC was expressed in%. ** p <0.001, ns not significant (t-test)

2 : Increased adhesion of psoriatic PMNC to Thy-1 decreases during anti-psoriatic treatment with Raptiva ^®

The adhesion of PMNC to Thy-1 and vector-transfected CHO cells and the disease activity (PASI) were before time dependent on seven patients with chronic plaque psoriasis and examined 2, 4, 8 and 12 weeks after the intialen Raptiva ^®. ** p <0.001, ns not significant (t-test). To illustrate the Thy-1 specific adhesion, the number of cells adhering to the vector-transfected control cells was subtracted from the cells adhering to Thy-1. The number of adherent healthy PMNC was set at 100% and the change in psoriatic PMNC adhesion compared to healthy PMNC was expressed in%.

3 : Adhesion psoriatic PMNC to ICAM-1 during an anti-psoriatic treatment with Raptiva ^®

The adhesion of PMNC to ICAM-1 and vector-transfected CHO cells and the disease activity (PASI) were before time dependent on seven patients with chronic plaque psoriasis and examined 2, 4, 8 and 12 weeks after the intialen Raptiva ^®. ** p <0.001, ns not significant (t-test). To illustrate the ICAM-1-specific adhesion, the number of cells adhering to the vector-transfected control cells was subtracted from the cells adhering to ICAM-1. The number of adherent healthy PMNC was set at 100% and the change in psoriatic PMNC adhesion compared to healthy PMNC was expressed in%.

4 Adhesion of neutrophil granulocytes to Thy-1 decreases with ciclosporin treatment

neutrophils Granulocytes (PMNC) from patients with psoriasis (n = 7) were present Start treatment with cyclosporine and after 2, 4, 8 and 12 weeks prepared for cyclosporin treatment and the adhesion investigated for Thy-1 and vector-transfected CHO cells. To the representation Thy-1 specific adhesion was the number of cells adhering to the vector-transfected control cells from those adhering to Thy-1 Cells removed. [* p <0.05]

5 : Thy-1-mediated adhesion of neutrophilic granulocytes correlates with disease activity (PASI)

neutrophils Granulocytes (PMNC) from patients with psoriasis (n = 7) were present Start treatment with cyclosporine and after 2, 4, 8 and 12 weeks the Ciclosporin treatment prepared and the adhesion investigated for Thy-1 and vector-transfected CHO cells. To the representation Thy-1 specific adhesion became the adhesion withdrawn to the vector-transfected control cells. Parallel became the disease activity (PASI) and the reduction of PASI in the course of treatment in% determined [* p <0.05 for Thy-1-mediated adhesion; # p <0.005 for PASI reduction]

6 : Direct inhibition of the adhesion of PMNC to Thy-1 and activated EC by ciclosporin.

PMNC of healthy controls and patients with psoriasis vulgaris (n = 5) were prepared and the adhesion Thy-1, vector-transfected CHO cells and human dermal microvascular EC (HDMEC) after incubation with cyclosporin for 30 min. To the representation Thy-1 specific adhesion became the adhesion withdrawn to the vector-transfected control cells. Adhesion of the PMNC without ciclosporin was set 100%. [* p <0.01]

Embodiment 1

Test system and test procedure

manufacturing a stably transfected with Thy-1 CHO cell line The Thy-1 cDNA (Accession number: M11749) from base 27 to 1519 containing the complete mature Thy-1 protein was encoded into the vector pcDNA6 (Invitrogen) cloned containing a blasticidin resistance gene. CHO cells were treated with 0.05% trypsin, 0.02% EDTA replaced and with 1 mg of Thy-1 containing plasmid or with the vector alone Electroporation transfected (300 V, 1500 mF, Equibio (Peglab, Erlangen, Germany)). The cells were immediately transferred to cell culture vessels and blasticidin (7.5 mg / ml, Invitrogen) was added after 48 hours, to select stably transfected cells. Transfected cells, which express Thy-1 were obstructed by flow cytometry Use of an anti-Thy-1 antibody (mABAS02) identified.

Transfected CHO cells were cultured in DMEM / RPMI 1640 (1: 1) with 10% fetal calf serum, 1% ascorbic acid and 1% penicillin / streptomycin at 37 ° C, 5% CO ₂ . The passage was carried out after reaching confluency using 0.05% trypsin and 0.02% EDTA.

cell adhesion assays

For cell adhesion assays, CHO cells transfected with Thy-1 or the control vector and PMA (20 ng / ml) stimulated HDMEC were used. The cells were seeded on chamber slides (NUNC, Wiesbaden, Germany) and incubated for 24 to 48 hours at 37 ° C and 5% CO ₂ . After reaching a confluent cell monolayer, the transfected cells were fixed by incubation with ice-cold methanol and stored at -20 ° C. HDMEC were used immediately after reaching a confluent cell monolayer in cell adhesion assays.

to Isolation of PMNC was done in whole blood with dextran M70 (2% final concentration; Sigma-Aldrich, Taufkirchen, Germany) for Incubated for 60 minutes at room temperature. The plasma was removed and PMNC were assayed by Ficoll density gradient centrifugation (lymphocytes Separation medium, r¼ 1,077 g / ml; MP-Biomedicals, Aurora, OH) at 800 x g for 20 minutes. The PMNC-containing pellet was incubated for 1 minute with distilled water to the lysing remaining erythrocytes. Eventually, the PMNC got twice washed with phosphate buffered saline (PBS). After the isolation were the peripheral PMNC with D-biotinoyl ε-aminocaproic acid hydroxysuccinimide ester (Roche Biochemicals, Mannheim, Germany) biotinylated according to the manufacturer's instructions.

After washing three times with PBS, 2 × 10 ^{5 of} the PMNC in RPMI 1640 was added to each well and incubated at 37 ° C for 30 minutes. Unbound cells were removed by multiple RPMI 1640 washes and adherent cells were fixed with ice-cold methanol for 10 minutes at -20 ° C, air dried and blocked by incubation with PBS / 1% BSA. Subsequently, adherent biotinylated cells were visualized by incubation with CY3-conjugated streptavidin (Beckman Coulter, 1: 500 in PBS / 1% BSA) for 60 minutes at room temperature and finally washed twice with PBS. The number of cells adherent to the confluent cell monolayer was counted twice independently in 10 microscopic fields (20x magnification). For each experiment, the average of both counts was calculated and used to perform the statistical analysis. In the two independent counts, there were no significant differences in the absolute number of adherent healthy and psoriatic PMNC, indicating that there is no significant interobserver variability (P¼0.85).

Subsequently was the adhesion examined by PMNC from different healthy donors. In the same Experiment, the adhesion was more healthy PMNC determined by four different donors. The standard deviation was low and the standard deviation error was less than 15%. On the basis of these results, the adhesion of PMNC became different healthy individuals (two per experiment) used as a control around the adhesion from psoriatic PMC and healthy PMNC.

The absolute number of adherents PMNC psoriatic or healthy donor was different from assay to assay. Therefore, the adhesion of "healthy PMNC" was set to 100% and the change the adhesion psoriatic PMNC versus healthy PMNC was expressed in%.

to Representation of Thy-1-specific adhesion was the number of the vector-transfected control cells adherent Cells from those attached to Thy-1 Cells removed.

Embodiment 2

Thy-1 mediated adhesion of neutrophilic granulocytes as a marker for successful treatment response of patients with psoriasis treated with Raptiva (efalizumab)

In The study included seven patients with moderate to severe chronic Plaque psoriasis included in other systemic therapies including Cyclosporine, methotrexate and PUVA had no effect or who have a contraindication or intolerance to these therapies.

From These patients and healthy donors were polymorphonuclear cells (PMNC, neutrophils) and the adhesion to Thy-1, ICAM-1 and Vector transfected control cells were used in static cell adhesion assays according to the embodiment 1 determined. To represent the Thy-1 or ICAM-1-specific adhesion was the number of adherent to the vector-transfected control cells Cells are subtracted from the cells adhering to Thy-1 and ICAM-1, respectively. The number adherent healthy PMNC was set to 100% and the change in psoriatic adhesion PMNC versus healthy PMNC was expressed in%.

The psoriatic PMNC adhered significantly more strongly to Thy-1 than healthy neutrophils ( 1 ). Although ICAM-1 was long considered to be the most important adhesion molecule on activated EC for mediating a stable adhesion of leukocytes, no significant differences were found between psoriatic PMNC and healthy PMNC upon adhesion to ICAM-1 transfected cells ( 1 ).

Neutrophil adhesion of psoriasis patients to Thy-1 or ICAM-1 was before the beginning of treatment with Raptiva ^® and 2, 4, 8 and 12 wk examined after the start of treatment. The adhesion of healthy donor neutrophils to Thy-1, ICAM-1 and vector-transfected control cells was detected in the same manner on the individual screening days.

The adhesion of neutrophils to Thy-1 was significantly (p <0.01) reduced two weeks after starting treatment with Raptiva ^® and almost reached the levels of healthy control PMNC ( 2 ). After 8 weeks of treatment, adhesion to Thy-1 further decreased to about 50% of that of healthy PMNC. In contrast, the adhesion of psoriatic PMNC to ICAM-1 was not enhanced compared to healthy PMNC. Adhesion to ICAM-1 was unchanged four weeks after initiation of treatment ( 3 ). After 8 weeks of treatment with Raptiva ^® adhesion psoriatic PMNC to ICAM-1 was distinctly down-regulated.

Therefore suitable only the Thy-1 mediated adhesion of neutrophils to determine early changes in the activation of neutrophils and thus is a more robust parameter to monitor the therapeutic efficacy of Raptiva ^® in psoriasis.

Subsequently, it was investigated whether the adhesion of neutrophils to Thy-1 or ICAM-1 as parameters for the activation of neutrophils and for the prediction of disease activity in psoriasis. The disease activity was determined in all patients by means of PASI (Psoriasis Area and Severity Index). Although ICAM-1 is an important adhesion molecule, there was no correlation between the ICAM-1-mediated neutrophil adhesion and disease activity during treatment with Raptiva ^® ( 3 ). In contrast, the adhesion of neutrophils to Thy-1 was down-regulated in parallel with disease activity (PASI). The adhesion of psoriatic neutrophils to Thy-1 was significantly downregulated already two weeks after initiation of treatment with Raptiva, at which time no significant changes in the PASI value could be detected. After another two weeks, however, the PASI value was significantly reduced. These data show that a decrease in the adhesion of psoriatic neutrophils to Thy-1 is the first indication of successful treatment even before clinical improvement can be observed.

Embodiment 3

Thy-1 mediated adhesion of neutrophilic granulocytes as a marker for successful treatment response of patients with psoriasis under treatment with cyclosporine

The adhesion of neutrophilic granulocytes from patients with psoriasis in the course therapy with cyclosporin to Thy-1 transfected CHO cells according to the embodiment 1 tracked.

To Become peripheral neutrophil granulocytes from 7 patients with psoriasis isolated. The patients had severe psoriasis and received cyclosporine in a dosage of 3-5 mg / kg body weight. Acceptance dates are before the start of therapy and during the course of therapy (2, 4 and 8 weeks). Subsequently will the adhesion neutrophil granulocytes to Thy-1 transfected CHO cells and control cells. In parallel, the disease activity by means of determined by the PASI. Finally becomes Thy-1-mediated adhesion and the improvement of the clinical picture (PASI reduction) compared.

The neutrophil granulocytes of patients with psoriasis adhere significantly more to Thy-1 than healthy granulocytes ( 1 ).

As in 4 As shown, Thy-1-mediated adhesion decreases significantly during the course of ciclosporin treatment. The data show that the anti-inflammatory effect of cyclosporine is also based on an influence on granulocyte functions.

As in 5 As shown, in parallel with the decrease in Thy-1-mediated adhesion, the clinical picture improves, which is reflected in the reduction of PASI as a marker of disease activity.

there could be found that a significant reduction of PASI in 4 of 4 patients also with a decrease in Thy-1-mediated adhesion at or below the level of neutrophilic granulocytes of healthy Donate within the first 2-4 Weeks after treatment was associated with cyclosporine. at Three patients were not significantly improved with cyclosporine achieved the clinical picture. In 2 of these 3 patients There was no significant decrease in Thy-1-mediated in vitro adhesion at or below the level of healthy neutrophilic granulocytes instead of.

These Data show that the Thy-1-mediated adhesion of neutrophilic granulocytes a marker for the response to anti-psoriatic ciclosporin therapy is. A reduction in Thy-1 adhesion of neutrophils of patients with psoriasis taking cyclosporine therapy An improvement in the clinical picture and an unchanged Thy-1 adhesion of neutrophils Granulocytes from patients with psoriasis taking cyclosporine therapy is associated with a treatment failure.

Embodiment 4

validation and application of a test system for in vitro analysis of anti-inflammatory Effect of drugs

PMNC of healthy controls and patients with psoriasis vulgaris (n = 5) were prepared and the adhesion Thy-1, vector-transfected CHO cells and human dermal microvascular EC (HDMEC) after incubation with cyclosporin A for 30 min.

Depending on the concentration, cyclosporin A is capable of inhibiting the adhesion of neutrophilic granulocytes from healthy donors as well as from patients with plaque psoriasis in vitro to Thy-1-transfected CHO cells ( 6A ) to inhibit.

The physiological relevance of these data is substantiated by the fact that cyclosporin A concentration-dependently inhibits the adhesion of PMNC to primary, activated Thy-1-expressing human dermal microvascular EC, the physiological target cells ( 6B ).

The Test system for in vitro analysis of pharmaceuticals is thus able to a potentially anti-inflammatory To indicate the effect of a drug.

Claims (21)

Test system that uses at least cells that are human Thy-1 (CD90) expressing contains. Test system according to claim 1, characterized that the cells expressing human Thy-1 (CD90) activated microvascular dermal endothelial cells, human dermal fibroblasts or a stable with the Thy-1 transfected CHO cell line. Test system according to claim 2, characterized that the stably transfected with Thy-1 CHO cell line is available by cloning the cDNA for Human Thy-1 (CD90) (Accession number: M11749) from nucleotide 27 to nucleotide 1519 in the expression vector pcDNA6, transfecting of CHO-K1 cells (ATCC CCL61) and selection of a stably expressing Cell line. Test system according to one of claims 1 to 3, characterized that the test system in addition Contain control cells for standardization. Test system according to one of claims 1 to 4, characterized that the cells expressing human Thy-1 (CD90) as a monolayer are fixed on a firm surface. Test system according to one of claims 1 to 5, characterized that the test system in addition Cells expressing a Thy-1 (CD90) ligand at the surface contains. Test system according to claim 6, characterized in that the cells expressing a Thy-1 (CD90) ligand on the surface are monocytes, neutrophilic granulocytes or melanoma cells or one with a Thy-1 ligands are transfected CHO cell line. Test system according to claim 7, characterized that the cells expressing a Thy-1 (CD90) ligand at the surface with Mac1 (CD11b / CD18) or aVb3 (CD51 / CD61) transfected CHO cell lines are. Use of a test system according to one of claims 1 to 5 for determining the efficacy of biologics and systemic Therapies in psoriasis therapy. Use of a test system according to one of claims 6 to 8 for the identification of substances with anti-inflammatory or anti-metastatic action. Method for determining the effectiveness of biologics and systemic therapies for psoriasis therapy with the following steps: a. Determining the cell-cell interaction of CHO cells, transfected with human Thy-1 (CD90) with granulocytes of patients with a biological or systemic therapy be treated b. Determine the cell-cell interaction of CHO cells transfected with human Thy-1 (CD90) with Control cells c. Comparing cell-cell interactions, in which - one Decreased cell-cell interaction of CHO cells with human Thy-1 (CD90) transfected are, with granulocytes of patients, with a biological or treated systemic therapy, an indication of the anti-inflammatory Effect of biologic or systemic therapy is and - not one Decreased cell-cell interaction of CHO cells with human Thy-1 (CD90) transfected with granulocytes from patients, who are treated with a biological or systemic therapy, an indication for Therapeutic gastric biologic or systemic therapy is. Method according to claim 11, characterized in that in order to determine the cell-cell interaction, the granulocytes are labeled become. A method according to claim 12, characterized in that as a marker Biotinylie carried out. Method according to claim 13, characterized in that that adherent Cells by a streptavidin-coupled fluorescent dye stained become. Method according to claim 11, characterized in that that determining the cell-cell interaction by means of automated Analysis is done. Method for the identification of substances with anti-inflammatory or anti-metastatic Effect with the steps a. Determine the cell-cell interaction of cells expressing human Thy-1 (CD90) on their surface, with cells expressing a Thy-1 (CD90) ligand at the surface, in the absence of the test substance, b. Determine the cell-cell interaction of cells expressing human Thy-1 (CD90) on their surface, with cells expressing a Thy-1 (CD90) ligand at the surface, in the presence of the test substance, c. Comparing cell-cell interactions, wherein a diminished in the presence of the test substance cell-cell interaction an indication for the anti-inflammatory or anti-metastatic effect of the test substance. Method according to claim 16, characterized in that that the test substance first either with the Thy-1 expressing cells or with the Thy-1 ligand expressing Cells is incubated before the process step b) is performed. Method according to claim 16, characterized in that that for determining the cell-cell interaction the cells which to express a Thy-1 (CD90) ligand on its surface. Method according to claim 18, characterized that a biotinylation is carried out as a label. Method according to claim 19, characterized that adherent Cells by a streptavidin-coupled fluorescent dye stained become. Method according to claim 16, characterized in that that determining the cell-cell interaction by means of automated Analysis is done.