JP2007051980A - Examination method of graft-versus-host disease, reagent for examination, and screening method of preventive and/or remedy - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an effective examination method independent of tissue biopsy and a reagent relative to GVHD after grafting, and a screening method of a preventive and/or a remedy having a reduced side effect which is not an immunosuppressive agent. <P>SOLUTION: It is found that fractalkine (Fractalkine/CX3CL1) which is one kind of chemokines is strongly expressed in a patient tissue of a GVHD crisis recipient, and that expression of acceptor CX3CR1 of the fractalkine is lowered in a CD14 positive monocyte. The GVHD after grafting can be examined by using the fractalkine and/or its acceptor CX3CR1 as a marker. A medicine for prevention and/or remedy to the GVHD can be screened by detecting fluctuation of expression intensity of the fractalkine and/or its acceptor CX3CR1. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a test method for graft versus host disease (hereinafter referred to as “GVHD”), a test reagent, and a screening method for a prophylactic and / or therapeutic drug after transplantation.

  Allogeneic hematopoietic stem cell transplantation is a treatment method that hopes to cure malignant hematopoietic tumors, other refractory blood diseases, and some non-blood refractory diseases, and is currently widely used around the world. There are various serious complications associated with allogeneic transplantation. Among them, chronic GVHD is difficult to control, and the lesions are spread to multiple organs, and the quality of life (QOL) after transplantation is significantly worsened. It is the main cause. It develops after about 100 days after transplantation, shows a pathological condition similar to autoimmune disease, and many deaths occur due to complications of infectious diseases. Inclusion of mild cases is reported to affect 40-80% of allograft recipients. Although trying to control the disease state with the administration of immunosuppressants, the effect is often low. In recent years, there have been cases where lung transplantation is performed due to respiratory failure due to chronic GVHD of the lung.

  In hematopoietic transplantation, siblings matching HLA, which is the major histocompatibility antigen of humans, are usually selected as donors. However, since transplanted hematopoietic cells contain T lymphocytes and their progenitors, so-called other than HLA When lymphocytes recognize non HLA antigens as targets, various transplantation immune reactions may occur. The unique symptom caused by this is clinically called GVHD.

  As preventive or therapeutic agents for GVHD in allogeneic hematopoietic stem cell transplantation, immunosuppressive agents such as steroids, cyclosporine (CsA), tacrolimus (FK506) are administered for a long time. In allogeneic hematopoietic stem cell transplantation, not only the neutropenic state by engraftment, but also a strong immunosuppressive therapy for GVHD delays the recovery of cellular immunity, causing problems such as opportunistic infection. In recent years, there are an increasing number of cases that are placed in a strong immunosuppressed state, so there have been reports of cases where the introduction of new antifungal agents is desired without the improvement of symptoms such as fever under the preventive administration of conventional antifungal agents (Non-Patent Document 1).

  If other drugs can be used to prevent or treat GVHD, side effects such as infection can be reduced and new therapeutic effects can be expected. However, for chronic GVHD, an excellent mouse model is available. However, the onset mechanism is almost unknown, so that no new drug has been developed.

  The diagnosis of GVHD is performed pathologically in each tissue such as skin biopsy, liver biopsy, lip biopsy. The initial inflammatory change in each diseased tissue is mainly lymphocyte infiltration into epithelial tissue, exocrine secretion, and endocrine gland, but as it progresses, fibrosis, stenosis, occlusion, and atrophy are observed. However, there have been few reports on diagnostic methods for GVHD that do not involve biopsy. Regardless of biopsy, if GVHD can be diagnosed before the symptoms progress, the burden on the patient will be reduced and a treatment plan will be easier.

  CX3CR1, which is a receptor for fractalkine / CX3CL1, which is a kind of chemokine, is expressed in lymphocytes and monocytes in general. Fractalkine or fractalkine receptor CX3CR1 knockout mice have been shown to be less susceptible to arterial atherosclerosis, where local infiltration of monocytes is considered to be the primary etiology It has been reported that fractalkine is an important factor for attracting monocytes locally in the pathological state (Non-Patent Documents 2 to 4). However, the relationship between fractalkine and post-transplant GVHD has not been reported.

Jpn. J. Med. Mycol. 2003; 44: 187-191 J. Exp Med. 2003; 197: 1701-1707 Mol. Cell Biol. 2001; 21: 3159-3165 J. Clin. Invest. 2003; 111: 333-340

  An object of the present invention is to provide an effective diagnostic method for GVHD irrespective of tissue biopsy. Furthermore, regarding GVHD, the present invention relates to a screening method for effective preventive and / or therapeutic agents with reduced side effects.

  In order to solve the above-mentioned problems, the inventors of the present invention focused on CD14-positive monocytes / macrophages, and particularly chemokines that attract them locally, and conducted extensive research. As a result, the expression rate of fractalkine receptor (CX3CR1) is decreased in peripheral blood monocytes (CD14 positive monocytes) of chronic GVHD developing recipients, and the strong expression of fractalkine in the tissues and CD14 positive cells As a result, the present invention was completed.

That is, this invention consists of the following.
1. A method for testing graft-versus-host disease, comprising detecting fractalkine and / or its receptor CX3CR1 as a marker.
2. 2. The method for examining graft-versus-host disease according to item 1 above, wherein the expression intensity of fractalkine on the tissue of a transplant recipient is detected.
3. 3. The method for examining graft-versus-host disease according to item 1 or 2, wherein the expression intensity of CX3CR1 on peripheral blood monocytes (CD14 positive cells) of a transplant recipient is detected.
4). 4. A test reagent for use in the method for testing graft-versus-host disease according to any one of items 1 to 3.
5. A method for screening a drug for prevention and / or treatment of graft-versus-host disease, wherein fractalkine and / or its receptor CX3CR1 is used as a marker, and variation in expression intensity of the marker is used as an index.
6). 6. The screening method according to item 5 above, wherein fluctuation of the expression intensity of fractalkine is used as an index.
7). 7. The screening method according to 5 or 6 above, wherein the change in the expression intensity of CX3CR1 on peripheral blood monocytes (CD14 positive cells) is used as an index.

According to the present invention, CX3CR1, which is a receptor for fractalkine on transplant recipients, can be collected without significant invasion in patient tissue, for example, fractalkine expression in skin, or on peripheral blood monocytes (CD14 positive cells). By detecting the decrease in the expression, it is possible to diagnose whether the transplant recipient develops GVHD or may develop it in the future.
Furthermore, it is possible to provide an effective prophylactic and / or therapeutic screening method for GVHD with fewer side effects than immunosuppressive agents used for the purpose of suppressing the action of lymphocytes.

  The transplant to which the present invention is applied is not particularly limited, and any transplant can be considered. Specific examples include tissues (skins such as skin, cornea and bone) or organs (liver, heart, kidney, lung, pancreas, etc.) and blood-related (hematopoietic stem cells, during lymphocyte infusion) transplantation, etc. It is applied to transplantation where the onset of GVHD can be a problem, and more preferably applied to blood-related transplantation.

  The prophylactic and / or therapeutic agent obtained by the test method, test reagent, and screening method of the present invention can be applied to either acute GVHD or chronic GVHD, but more preferably chronic GVHD.

  For example, fractalkine is strongly expressed in normal lung tissue but only weakly expressed in normal skin. Although the expression level of fractalkine in lung tissues of chronic GVHD developing recipients is almost the same as that in normal lung tissues, it was observed that it is strongly expressed in the skin of chronic GVHD developing recipients (FIGS. 3-1, FIG. 3). 4-1). Expression of fractalkine was also observed in the skin of a recipient with acute GVHD, but only in a part of the site exhibiting the characteristics of acute GVHD. In comparison with this, in the case of chronic GVHD, expression of fractalkine was observed in a wider range.

  In the case of healthy individuals, expression of CX3CR1, which is a receptor for fractalkine, is observed on peripheral blood monocytes (CD14-positive cells), but CX3CR1 of chronic GVHD developing recipients has a considerably low expression level. . On the other hand, the expression level of CX3CR1 in transplant recipients who are not chronic GVHD is almost the same as that in healthy individuals (see FIG. 1). On the other hand, in lymphocytes, there is no correlation regarding the expression of CX3CR1 between recipients who develop chronic GVHD and recipients who do not develop chronic GVHD (see FIG. 2). From this, variation in the expression intensity of CX3CR1 on peripheral blood monocytes (CD14 positive cells) can be a marker for the development of chronic GVHD.

  From the above, the present invention is more preferably applied to the case of chronic GVHD. Here, the scoring system which becomes a standard of the severity of chronic GVHD is shown below.

Chronic graft-versus-host disease (severity classification)
(General general condition)
0: No symptoms (0 by ECOG classification, ECOG: Eeastern Cooperative Oncology group)
1: Symptoms (1 for ECOG classification)
2: Symptoms (2 for ECOG classification)
3: Symptoms (3 by ECOG classification)

(Skin)
0: No skin symptoms due to chronic graft-versus-host disease.
1: Skin rash or skin hardening less than 18% of body surface area.
2: Skin rash or sclerosis of 18-50% of body surface area.
3: Skin rash or skin hardening exceeding 50% of the body surface area.

(mouth)
0: No symptom, no physics
1: Oral symptoms (dryness, abnormal taste, pain in the oral cavity) or findings (redness, hyperkeratosis, lichenification or ulceration).
2: Oral symptoms or findings that partially prevent oral intake.
3: Oral symptoms or findings that largely prevent oral intake.

(eye)
0: no symptoms, no keratitis 1: asymptomatic keratitis, or mild dryness (use of eye drops for moisturizing up to 3 times a day).
2: Symptoms due to keratoconjunctivitis but no visual impairment, or instillation of 4 or more times a day is necessary for moisturizing
3: Vision loss due to corneal ulcer with pseudomembranization, or ligation of nasolacrimal duct, special glasses required to reduce pain and discomfort are required, or work is impossible due to eye symptoms alone is there.

(Digestive organ)
0: No digestive symptoms 1: Although there are digestive symptoms, intravenous fluid replacement and nutrition are not required.
2: There are digestive symptoms and partially requires intravenous fluid replacement and nutritional support.
3: Has gastrointestinal symptoms and is totally dependent on parenteral nutrition, or> 15% weight loss or esophageal dilatation.

(liver)
0: Total bilirubin and alkaline phosphatase ≦ 1 × reference value.
1: Total bilirubin or alkaline phosphatase> 1 × reference value and ≦ 2 × reference value 2: Total bilirubin or alkaline phosphatase> 2 × reference value and ≦ 4 × reference value 3: Total bilirubin or alkaline phosphatase> 4 × reference value, AST , ALT> 3 × reference value

(lung)
0: No symptoms 1: 51-57% of normal FEV1.0, <65% of normal FEV1.0 / FVC, or 12% or more decrease in DLCO per year, or difficulty breathing during moderate exercise.
2: 25 to 50% of normal FEV1.0, breathing difficulty during light exercise, or decrease in oxygen saturation during exercise.
3: FEV1.0 <25% normal, breathing difficulty at rest, or requires oxygen inhalation during exercise or rest.

(Joint and fascia)
0: No range of motion restriction 1: Movement restriction limited to arms or legs.
2: Contracture that does not affect ADL (activity in daily life), or movement limitation that affects both upper and lower limbs.
3: Contracture that reduces ADL (activity in daily life).

(Genital)
0: No symptoms 1: ADL (activity in daily life) or symptoms that do not affect intercourse (atrophy, redness, lichenification, edema)
2: Symptoms that do not affect ADL (activity in daily life) but cause sexual difficulties.
3: Symptoms affecting ADL (activity in daily life).

The present invention provides a method for testing GVHD by detecting fractalkine and / or its receptor CX3CR1 as a marker.
For example, the onset of GVHD can be examined by examining the expression level of fractalkine in the patient tissue after transplantation in the present invention, particularly preferably skin tissue. The expression level of fractalkine can be determined by a method known per se, and a tissue cell obtained can be detected by staining or the like. In addition, any means for examining the expression level of fractalkine developed in the future can be employed. For example, it can be examined by examining the expression of a fractalkine-specific gene.

  As another test method, GVHD can be tested by collecting peripheral blood from a transplant recipient and detecting CX3CR1 expressed in CD14-positive monocytes in the peripheral blood. Periodic collection of CD14-positive monocytes in peripheral blood and observation of changes in CX3CR1 makes it possible to predict the onset of GVHD without biopsy, and to plan treatment early It is. Observation of the variation of CX3CR1 can be performed by a method known per se, for example, a flow cytometry method or any method developed in the future can be adopted. For example, it is possible to test by examining the expression of a gene specific to CX3CR1.

  The present invention also extends to reagents that can be used in the above-described inspection methods. For example, a probe, primer, or the like that can detect fractalkine and / or CX3CR1 antigens, antibodies, or genes related to these proteins can be provided as reagents.

  The present invention also relates to a method for screening a drug for prevention and / or treatment of GVHD, characterized by using fractalkine and / or its receptor CX3CR1 as a marker, and using the change in expression intensity of the marker as an index. It extends. In the present invention, the relationship between post-transplant GVHD and fractalkine, its receptor CX3CR1, was found for the first time. This suggests that the mechanism that fluctuates the expression intensity of fractalkine and / or its receptor CX3CR1 is related to the development of GVHD after transplantation. Therefore, by using fractalkine and / or its receptor CX3CR1 as a marker and selecting a substance that can change the expression intensity of the marker from candidate compounds and candidate substances, the onset of GVHD after transplantation can be prevented or treated. The substance can be selected.

  Conventionally, there is no effective treatment method for post-transplantation GVHD, particularly chronic GVHD, and administration of immunosuppressive agents used for the purpose of suppressing the action of lymphocytes has been the mainstream. However, in the present invention, since there was no correlation between the expression level of CX3CR1 on lymphocytes and chronic GVHD, administration of immunosuppressive agents used for the purpose of suppressing lymphocyte action prevented chronic GVHD and It has been suggested that the treatment cannot be fully effective. By using a drug according to the onset mechanism specific to post-transplantation GVHD, particularly chronic GVHD, side effects are reduced, and effective preventive and therapeutic effects can be obtained.

  EXAMPLES The present invention will be described below with reference to examples, but it is obvious that the present invention is not limited to these examples.

(Example 1) Analysis by flow cytometry 1) Blood sample Peripheral blood was collected from 17 recipients after allogeneic hematopoietic stem cell transplantation and expressed on the surface of CD14-positive monocytes and lymphocytes using flow cytometry. The expression intensity of CX3CR1 (fractalkine receptor) was examined.
Each recipient had 8 males, 9 females, 20-64 years of age, median of 45 years, 97-2786 days after transplantation, and median of 681 days.

2) Clinical evaluation Clinical symptoms (severity) of chronic GVHD were evaluated by changing "GVHD Morbidity Assessment". Each recipient's symptoms were evaluated according to the scoring system described above, and the sum of each score was used as the chronic GVHD (cGVHD) score.

3) Flow cytometry Blood samples were stained with PE-conjugated anti-human CD14 antibody (CALTAG) along with FITC-conjugated anti-human CX3CR1 antibody (MBL) or FITC-conjugated isotype matched control (MBL), followed by Red blood cells were lysed with a lysis solution (BD Pharmingen). Analysis was performed on a FACSCalibur flow cytometer equipped with CellQuest software (Becton Dickinson).

4) Results The results of flow cytometry for CD14 positive monocytes are shown in FIG. 1-1. An isotype control peak was observed on the left side, and a CX3CR1 peak was observed on the right side. In recipients with strong chronic GVHD symptoms, the expression intensity of CX3CR1 in CD14 positive monocytes was weaker than in recipients with chronic GVHD symptoms.
FIG. 1-2 shows the correlation between the chronic GVHD score and the value obtained by subtracting the control value by quantifying the CX3CR1 fluorescence intensity of CD14 positive monocytes. Correlation was determined using the Spearman rank correlation coefficient. P <0.05 was considered statistically significant. As a result, CX3CR1 expression intensity on peripheral blood CD14-positive monocytes was lower in recipients with a statistically significant difference and stronger chronic GVHD symptoms.
The results of flow cytometry for lymphocytes are shown in FIG. 2-1. Further, FIG. 2-2 shows the correlation between the chronic GVHD score and the value obtained by subtracting the control value by digitizing the CX3CR1 fluorescence intensity of lymphocytes. As a result, there was no correlation between the degree of chronic GVHD symptoms and the expression level of CX3CR1 in lymphocytes.

(Example 2) Tissue immunoassay 1) Tissue samples Skin biopsy specimens were collected from four patients who developed skin rash due to chronic GVHD among recipients after transplantation of allogeneic hematopoietic stem cells. In addition, because of severe respiratory failure due to obstructive bronchiolitis (BO) after transplantation of allogeneic hematopoietic stem cells, the lung tissue was removed from 4 patients who had undergone lung transplantation. Collected. Normal skin and lung tissue was purchased from Biochain Institiute.
The protocol is approved by Okayama University's Internal Review Board, and informed consent is obtained from patients.
Fractralcaine and CD14 positive cells expressed on these tissues were examined.

2) Tissue immunoassay Formalin-fixed and paraffin-embedded specimens were sliced to a thickness of 5 μm, deparaffinized, and antigen recovered by microwave in citrate buffer.
Samples were soaked in 3% H2O2 to eliminate endogenous peroxidase, then blocked with phosphate buffer (PBS) + 5% horse serum, polyclonal goat anti-human fractalkine antibody (R & D systems) or control goat IgG (Sigma Followed by biotinylated horse anti-goat IgG (Vector Laboratories). The staining signal was enhanced using an avidin-biotin peroxidase complex (ABC) kit (Vector Laboratories). Antigens were visualized with VIP (Vector Laboratories). For CD14 staining, samples were blocked with PBS + 5% sheep serum and incubated with biotinylated sheep anti-human CD14 antibody (R & D systems) or biotinylated control sheep IgG (Sigma). Staining was visualized using ABC kit (Vector Laboratories) and VIP (Vector Laboratories). Methyl green (Vector Laboratories) was used for counterstaining. Control staining with non-specific antibodies was performed in all staining experiments, but no significant non-specific staining was seen.

3) Results The lung tissues of normal and chronic GVHD developing recipients are shown in FIGS. As a result, fractalkine was observed in the bronchiole epithelium in normal and chronic GVHD lung tissues, and was observed to be strongly expressed in all tissues (FIGS. 3-1, A, B). On the other hand, CD14 antigen was hardly stained in normal lung tissue, and CD14 antigen was observed to be scattered (FIG. 3-2, C), whereas it was wide in lung tissue of chronic GVHD developing recipients. The area was strongly stained and CD14 antigen was observed (Fig. 3-2, D). Therefore, it is considered that soluble fractalkine was released from bronchiolar epithelial cells of lung tissue and CD14 positive monocytes were called in the vicinity of lung tissue in recipients with chronic GVHD.

  The skin tissues of normal and chronic GVHD developing recipients are shown in FIGS. As a result, fractalkine was thinly stained in normal skin and only weak expression was observed (FIGS. 4-1, E), whereas in chronic GVHD developing recipients, staining was observed in a wide area, and epithelial It was observed that it was strongly expressed with thickening (Fig. 4-1, F). Keratinocyte proliferation was also observed in recipients with chronic GVHD. The CD14 antigen was hardly stained in normal skin and was hardly observed (Fig. 4-2, G), whereas the skin of recipients with chronic GVHD developed strong staining in the dermis, and in some cases CD14 antigen was also observed in the epidermis (FIG. 4-2, H). From this, it was considered that monocytes highly expressing CX3CR1 circulated and attracted to the dermis by fractalkine supplied from the epidermis of chronic GVHD-onset recipients.

As described above, a method for examining GVHD can be provided by detecting fractalkine and / or its receptor CX3CR1 as a marker. Furthermore, the present invention provides a method for screening a drug for the prevention and / or treatment of GVHD, characterized by using fractalkine and / or its receptor CX3CR1 as a marker and using the expression intensity variation of the marker as an index. it can.
This makes it possible to diagnose whether the transplant recipient has GVHD or can develop in the future. Compared to conventional biopsy diagnostic methods, the burden on the patient is reduced, effective diagnosis can be made, and an effective treatment plan can be made early.
In addition, the drug selected by the screening method of the present invention provides an effective prophylactic and / or therapeutic drug for GVHD with fewer side effects compared to immunosuppressive drugs used for the purpose of suppressing the action of lymphocytes. Can be provided.

It is a figure which shows the result of having investigated the CX3CR1 expression level in CD14 positive monocyte by flow cytometry. Example 1 It is a figure which shows the result of the correlation of a chronic GVHD score and CX3CR1 fluorescence intensity of a CD14 positive monocyte. Example 1 It is a figure which shows the result of having investigated the expression level of CX3CR1 in a lymphocyte by flow cytometry. Example 1 It is a figure which shows the result of the correlation of a chronic GVHD score and the CX3CR1 fluorescence intensity of a lymphocyte. Example 1 It is the figure which investigated the expression of fractalkine in the lung tissue of a normal and chronic GVHD (cGVHD) onset recipient. (Example 2) It is the figure which investigated the CD14 antigen in the lung tissue of a normal and chronic GVHD (cGVHD) onset recipient by the dyeing | staining. (Example 2) It is the figure which investigated the expression of fractalkine in the skin tissue of a normal and chronic GVHD (cGVHD) onset recipient by the dyeing | staining. (Example 2) It is the figure which investigated the CD14 antigen in the skin tissue of a normal and chronic GVHD (cGVHD) onset recipient by the dyeing | staining. (Example 2)

Claims (7)

A method for testing graft-versus-host disease, comprising detecting fractalkine and / or its receptor CX3CR1 as a marker. The method for examining graft-versus-host disease according to claim 1, wherein the expression intensity of fractalkine on the tissue of a transplant recipient is detected. The method for examining graft-versus-host disease according to claim 1 or 2, wherein the expression intensity of CX3CR1 on peripheral blood monocytes (CD14 positive cells) of a transplant recipient is detected. The test reagent used for the test method of graft-versus-host disease of any one of Claims 1-3. A method for screening a drug for prevention and / or treatment of graft-versus-host disease, wherein fractalkine and / or its receptor CX3CR1 is used as a marker, and variation in expression intensity of the marker is used as an index. 6. The screening method according to claim 5, wherein fluctuation of the expression intensity of fractalkine is used as an index. The screening method according to claim 5 or 6, wherein fluctuations in the expression intensity of CX3CR1 on peripheral blood monocytes (CD14 positive cells) are used as an index.