JP2006177679A - Diagnostic marker, medicinal effect marker and utilization method of them - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a diagnostic marker having high sensitivity and specificity and capable of diagnosing rejection reaction simply and noninvasively, and a biomarker as a medicinal effect marker of an immunosuppression agent. <P>SOLUTION: The concentration of lysozyme in blood or intestinal juice is measured, after organ transplantation with respect to a vertebrate and compared with the predetermined diagnostic reference value of the concentration of lysozyme that does not show rejection reaction. Further, the immunosuppression agent is administered to the vertebrate subjected to transplant surgery to compare the concentration of lysozyme with the diagnostic reference value. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a diagnostic marker and a drug efficacy marker characterized by being lysozyme, and methods for using them.

  The small intestine is said to be difficult to control rejection especially after transplantation because the organ itself has an important immune function. Currently, biopsy and endoscopy are performed as methods for diagnosing the degree of rejection after small intestine transplantation, but histological diagnosis by biopsy is highly invasive and takes time. The diagnostic accuracy is not sufficient, and in the case of partial rejection, the diagnosis may differ depending on the collected tissue. The problem is that inspection with a magnifying endoscope cannot be performed after the stoma is closed.

  Thus, citrulline in serum (for example, see Non-patent Document 1) and bile acid (for example, see Non-Patent Document 2) have been developed as non-invasive biomarkers for acute rejection, but sensitivity and specificity have been developed. Was not enough.

Recently, MRP8 / 14 complex (migration inhibitory factor (MIF) -related protein 8/14 complex) can be a biomarker for detecting inflammation associated with rejection of transplanted tissues of the kidney and small intestine. Although it was shown, this is an early marker at the time of inflammation, and rejection specificity has not been proved yet (see, for example, Non-Patent Document 3).
Pappas PA, et al. Transplantation 2001; 72: 1212-6 Miyagi K, et al. Transplant Proc 2000; 32: 1265-6 Ikemoto M, et al. Clinical Chemistry 2003; 49: 594-600

  Therefore, an object of the present invention is to provide a biomarker that has high sensitivity and specificity, and that can easily and non-invasively diagnose rejection. In addition, if a biomarker that can easily and non-invasively diagnose rejection is used, the efficacy of the immunosuppressant can be quickly evaluated when administered, so this biomarker can be used as a drug marker for immunosuppressants. Can also be used.

  In order to solve the above-mentioned problems, the present inventors screened a protein specifically expressed only in individuals in a group showing rejection by using a plasma of a rat individual after transplantation of a small intestine and using a protein chip system. Thus, a protein detected at a high concentration only in the rejection group was identified, and it was confirmed that the protein was lysozyme, and the present invention was completed.

  The diagnostic marker for rejection according to the present invention and the medicinal marker of an immunosuppressant are lysozyme. The rejection is preferably a rejection that occurs after small intestine transplantation.

  The method for diagnosing the degree of rejection and the method for evaluating the efficacy of an immunosuppressant according to the present invention include measuring the amount of lysozyme in blood or intestinal fluid in humans and non-human vertebrates. The vertebrate is preferably transplanted into the small intestine.

  The rejection diagnosis kit and immunosuppressive drug evaluation kit according to the present invention include an anti-lysozyme antibody.

  The screening method for an effective immunosuppressive agent according to the present invention is a screening method for vertebrates that require administration of an immunosuppressive agent, and is characterized by using an immunosuppressive drug efficacy marker that is lysozyme. In this screening method, in each of a plurality of individuals belonging to the vertebrate, blood or intestinal fluid is collected from the individual before and after administration of a different immunosuppressant, and the lysozyme concentration in the blood or intestinal fluid is measured, Comparing changes in lysozyme concentration in blood or intestinal fluid collected from the individual before and after administration of the immunosuppressive agent for different immunosuppressive agents may be included. The individual is preferably transplanted into the small intestine.

  In addition, the method for determining the dose of an appropriate amount of an immunosuppressive agent according to the present invention is a method in a vertebrate individual that requires administration of an immunosuppressive agent, and uses a medicinal efficacy marker of the immunosuppressive agent that is lysozyme. It is characterized by that. In this screening method, blood or intestinal fluid is collected from the vertebrate, lysozyme concentration in the blood or intestinal fluid is measured, and the amount of the immunosuppressant used in advance and the lysozyme concentration in the blood or intestinal fluid are prepared. The dose of the immunosuppressive agent necessary for reducing the lysozyme concentration in the blood or intestinal fluid of the vertebrate to a predetermined expression level is determined using a calibration curve showing the above correlation. The vertebrate is preferably transplanted into the small intestine.

  The method for diagnosing rejection according to the present invention includes measuring the amount of at least one of MRP8 and MRP14 in blood or intestinal fluid and the amount of lysozyme in vertebrates other than humans.

  In addition, the method for evaluating the efficacy of the immunosuppressive agent according to the present invention comprises the steps of: lysozyme in an amount of at least one of MRP8 and MRP14 in blood or intestinal fluid in a vertebrate other than a human administered with an immunosuppressant; And measure the amount.

  The rejection diagnosis kit and the immunosuppressive drug efficacy evaluation kit according to the present invention include at least one of an anti-MRP8 antibody and an anti-MRP14 antibody, and an anti-lysozyme antibody.

  ADVANTAGE OF THE INVENTION According to this invention, the sensitivity and specificity are high, and the biomarker as a diagnostic marker which can diagnose a rejection reaction simply and noninvasively and the medicinal efficacy marker of an immunosuppressive agent can be provided.

  Hereinafter, embodiments of the present invention completed based on the above knowledge will be described in detail with reference to examples. Unless otherwise stated in the embodiments and examples, J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Standard Protocols in Molecular Biology, John Wiley & Sons Ltd. The method described in the protocol collection, or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.

  The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention and are shown for illustration or explanation, and the present invention is not limited to them. It is not limited. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

== Lysozyme as a biomarker ==
In the present specification, lysozyme (EC 3.2.1.17) means an enzyme that hydrolyzes the β-1,4 bond between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc). In particular, lysozyme expressed in macrophages is preferable, and for example, lysozyme C widely distributed in the living world such as avian egg white, mammals, fish and insects is preferable. In the following examples, rat lysozyme C, Lysozyme C, type 1 (LYC_RAT), was the detection target.

  In vertebrate organ transplantation, the concentration of lysozyme in plasma increases during rejection, and the increase in plasma lysozyme concentration is suppressed by administration of an immunosuppressant, so in humans and non-human vertebrates, Lysozyme in plasma is useful as a biomarker such as a diagnostic marker for rejection after organ transplantation and a drug efficacy marker for immunosuppressants.

  Lysozyme is secreted from panate cells in the small intestine and is responsible for innate immunity (Mason & Taylor, J. Clin Pathol 28, 124-132, 1975). However, lysozyme is known to be produced and secreted from monocytes and macrophage cells. In Crohn's disease of inflammatory bowel disease, there is a marked increase in lysozyme in the blood, which has been shown to be due to macrophage exudation to the inflamed site of the intestine (Stamp GWH et al., Gastroenterology 103, 532-538, 1992). For this reason, it has been suggested that the increase in blood lysozyme concentration during rejection in organ transplantation is due to macrophages that have exuded into the transplanted organ. Therefore, regarding the transplanted organ, although the small intestine can be mentioned as a suitable transplanted organ, the organ is not limited to the small intestine as long as the macrophage exudes when rejection occurs.

  If such a biomarker in blood or intestinal fluid is used, blood or intestinal fluid can be used as a sample, so that the concentration of the biomarker can be measured periodically and simply. In addition, blood samples or intestinal fluid samples can be easily obtained because they are less invasive than needle biopsy and do not require advanced techniques such as needle biopsy.

== Lysozyme as a diagnostic marker for rejection ==
The following describes in detail how to use lysozyme as a diagnostic marker.

  Measurement of lysozyme concentration in human and non-human vertebrates after transplantation of organs such as the small intestine by Western blotting or ELISA using polyclonal or monoclonal antibodies specific to lysozyme in blood or intestinal fluid However, the measurement of lysozyme concentration is not limited to these methods, and various other methods such as RIA (radioimmunoassay) and EIA (enzyme immunoassay) are available. You may measure using. Hereinafter, an example of measurement of lysozyme concentration in blood will be described.

  First, a serum fraction is obtained by centrifuging blood collected from a vertebrate transplanted with an organ. Thereafter, serum is put into the well of an ELISA plate (for example, one using a 96-well plate) to which the anti-lysozyme antibody is adsorbed, and reacted with the anti-lysozyme antibody fixed to the plate. After removing the serum, the well is washed. Subsequently, the well is washed after reacting with a primary antibody (anti-lysozyme antibody) and a secondary antibody (for example, an anti-immunoglobulin antibody labeled with an enzyme). Color is developed using a substrate for the enzyme, the absorbance is measured with a microplate reader, and the lysozyme concentration is measured using a calibration curve prepared in advance. Examples of enzymes used as labels for secondary antibodies include, but are not limited to, horseradish-derived peroxidase (HRP), alkaline phosphatase, and the like. The calibration curve is prepared by performing the above reaction simultaneously on the serially diluted lysozyme solution and graphing the relationship between concentration and absorbance.

  In addition, as a diagnostic marker for rejection, not only the lysozyme concentration in blood or intestinal fluid but also the concentration of at least one of MRP8 (MIF-related protein 8) and MRP14 (MIF-related protein 14) is measured. The result may be used for diagnosis. Since MRP8 / 14 is an early marker of inflammation at the time of rejection, its characteristic as a marker is complementary to lysozyme, and by examining both markers, it is possible to reliably diagnose rejection from earlier. Become.

  The measurement of the concentration of MRP8 or MRP14 is preferably performed by ELISA using an antibody specific to them as in the case of lysozyme, but the measurement method is not particularly limited.

  When such a diagnostic marker for rejection is used, for example, the concentration of the diagnostic marker in blood or intestinal fluid collected from a vertebrate transplanted with the small intestine is periodically measured, and when a predetermined rejection is not caused. By comparing with the diagnostic reference value, it is possible to easily diagnose rejection.

  Regarding the diagnostic reference value, if factors other than rejection do not affect the diagnostic marker concentration, for example, the normal value of the diagnostic marker concentration before transplantation is used as the diagnostic reference value and compared with that value. The degree of reaction can be evaluated. However, if a factor other than rejection, for example, transplantation operation itself, affects the concentration of the diagnostic marker, transplantation is not performed in advance on the same kind of vertebrate as the vertebrate to be diagnosed. Surgery is performed according to the procedure, the correlation between the degree of rejection and the concentration of the diagnostic marker is examined, and the concentration of the diagnostic marker is associated with the degree of rejection. In those individuals, if the concentration of the diagnostic marker is higher than the value (normal value) of an individual who does not undergo transplantation or surgery, an individual who has not caused rejection after the transplantation operation (an individual in which transplantation itself has not been performed, The diagnostic marker concentration of an immunosuppressive agent or the like in which no rejection has occurred may be used as the diagnostic reference value.

  Then, the degree of rejection of the animal is evaluated from the concentration of the diagnostic marker of the individual who has undergone surgery, which is the actual diagnosis target. That is, if the concentration of the diagnostic marker of the subject individual is a diagnostic reference value, it is evaluated that rejection has not occurred, and the larger the diagnostic reference value, the greater the rejection is evaluated.

  In the case of humans, rejection of the transplanted surgery patient is evaluated by a method other than the method of the present invention, such as another biomarker, histological diagnosis, or endoscopic diagnosis. The diagnostic reference value is determined by measuring the concentration of the biomarker, examining the correlation thereof, and associating the concentration of the diagnostic marker with the degree of rejection in advance. In this case, it is preferable to evaluate rejection by using as many methods as possible or analyzing them from multiple aspects.

== Lysozyme as a drug efficacy marker for immunosuppressants ==
An immunosuppressive agent is administered to a vertebrate transplanted with an organ, and the degree of rejection inhibition, that is, the efficacy of the immunosuppressive agent can be evaluated from the concentration of lysozyme in blood or intestinal fluid.

  First, as described above, a diagnostic reference value is set in advance. Next, after organ transplantation to the subject individual, the lysozyme concentration in blood or intestinal fluid is measured by ELISA or the like as described above while administering an immunosuppressant. The efficacy of the immunosuppressive agent is evaluated by comparing the obtained value with a diagnostic reference value.

  By using this method for evaluating the efficacy of immunosuppressive agents, screening of effective immunosuppressive agents and determination of the dose of an appropriate amount of immunosuppressive agent can be performed. It is possible to select an immunosuppressive agent with little or its administration method.

  For example, in each vertebrate individual transplanted with an organ, blood or intestinal fluid is collected before and after administration of a different immunosuppressant, and the lysozyme concentration therein is measured. By comparing the lysozyme concentrations before and after the administration of the immunosuppressive agent, screening can be performed to identify the immunosuppressive agent having the lowest lysozyme concentration.

  In addition, blood or intestinal fluid is collected from vertebrate individuals transplanted into organs, the concentration of lysozyme in the sample is measured, and the correlation between the amount of immunosuppressant used in advance and the concentration of lysozyme in blood or intestinal fluid is shown. The calibration curve shown can be used to determine the dose of immunosuppressant necessary to reduce the lysozyme concentration in blood or intestinal fluid to a predetermined expression level. If the degree of rejection is defined in advance by the expression level of lysozyme, the dose of an appropriate amount of immunosuppressive agent can be determined in this way.

== Kit ==
In order for anyone to easily use the biomarker, it is preferable to commercialize it as a kit. The kit includes an anti-lysozyme antibody for detecting at least lysozyme in blood or intestinal fluid. Moreover, in order to improve diagnostic accuracy, one or both of anti-MRP8 antibody and anti-MRP14 antibody may be included.

(1) Preparation of rat ectopic small intestine transplantation model In this example, a rat ectopic small intestine transplantation model was prepared as a model animal for small intestine transplantation in vertebrates such as humans. Brown Norway (BN) and Lewis (LEW) rats weighing 200-350 grams were used and the experimental groups were divided into the following 4 groups (each group n = 5): Control group (Sham group) in which LEW rats were given only laparotomy; Group II. Allogeneic syngeneic transplant group from LEW rat to LEW rat (Syngeneic group); Group III. Allogeneic transplant group from BN rat to LEW rat (Allogeneic group or Allo group); Group IV. After allogeneic transplantation from BN rats to LEW rats, FK506 (0.64 mg / kg / day, im) was administered daily (Allo + FK group).

  Rat ectopic small intestine transplantation was performed using a stereomicroscope and a partial modification of the Monchik and Russell method (Surgery 70, 693-702, 1971) under Frocene anesthesia. Regarding donor operation, first, a laparotomy was performed with a midline incision, and then the mesenteric artery and vein to the large intestine were ligated and separated. After severing the tritium ligament, the splenic and gastric veins were peeled off and ligated and separated. The portal vein was stripped and exposed, releasing the duodenum from the pancreas. The superior mesenteric artery was ligated and disconnected on the peripheral side, and the terminal ileum was cut from the cecum. After ligation of the right renal artery, the abdominal aorta was exfoliated from the celiac artery bifurcation to the left renal artery bifurcation. 0.5 ml of heparinized saline was injected through the penile dorsal vein. Two 6-0 silk threads were passed through the superior mesenteric artery bifurcation of the abdominal aorta, and only the caudal side was ligated. The proximal side of the superior mesenteric artery was ligated on the distal side of the celiac artery bifurcation of the abdominal aorta. A 27G injection needle was punctured into the abdominal aorta, and 5 ml of heparinized physiological saline was injected to perfuse until the graft small intestine became white. The portal vein was cut off at the hepatic portal area. The abdominal aorta was ligated with 6-0 silk thread and dissected on the distal side of the ligature. The abdominal aorta was dissected proximally from the superior mesenteric artery bifurcation, and the graft small intestine was removed. The excised small intestine was washed and removed with intestinal contents with a physiological saline solution at 4 ° C., and then cold-stored in the same solution. Recipient manipulation was performed immediately after graft removal. For vascular anastomosis, the donor abdominal aorta and recipient abdominal aorta were anastomosed using 8-0 prolean thread. The vein anastomosed the donor portal vein to the recipient's inferior aorta. Both ends of the graft intestine were fixed to the abdominal wall as a stoma.

(2) Histological determination of rejection reaction Regarding the prepared rat ectopic small intestine transplantation model, on the 5th, 7th and 10th day after the operation, tissue sections of the transplanted part were prepared and stained with hematoxylin-eosin (HE). The determination was made according to a report by Todo et al. (Ann. Surg. 216, 223-234, 1992). The results are shown in FIG.

  In the non-rejection group and the immunosuppressant administration group, no rejection was observed even after 10 days from the operation, whereas in the rejection group, a weak rejection was observed on the fifth day after the operation, and the rejection gradually became stronger. Thus, on the 10th day after the operation, a very strong rejection was observed.

(3) Protein chip assay In this example, a plasma sample was obtained from the rat and the lysozyme concentration was measured using a protein chip.

First, 30 μl of urea buffer (PBS pH 7.4 containing 8M urea and 1% CHAPS) was added to 20 μl of plasma, left on ice for 15 minutes, and then diluted 20 times with PBS. The mixture was reacted for 1 hour on an IMAC chip activated with 50 mM NiSO ₄ and then washed with 400 μl of distilled water. After air drying, after addition of a matrix (sinapinic acid), measurement was performed using a ProteinChip Reader (Model PBSII, Ciphergen Biosystems), and the results were analyzed with ProteinChip Software 3.0 (Ciphergen Biosystems). The results are shown in FIG.

  In the Sham group, the lysozyme concentration hardly increased until the 10th day, but in the Syngeneic group, the Allo + FK group, and the Allo group, an increase in concentration was observed from the first day after the operation. However, after 5 days, a rapid concentration increase was observed only in the Allo group, corresponding to the histological determination results described above. The increase in lysozyme concentration in the Syngeneic group and Allo + FK group is thought to be due to the surgery itself. Therefore, in this example, when an immunosuppressive agent is given and the peak intensity decreases to about 5, it is considered that rejection is suppressed, and this result is also consistent with the histological determination result.

(4) Diagnostic marker for rejection and medicinal efficacy marker for immunosuppressant As described above, after small intestine transplantation, the lysozyme concentration in the blood increases corresponding to the intensity of histologically detected rejection, and immunosuppression When a drug is administered to suppress rejection, an increase in lysozyme concentration in blood is suppressed accordingly. Therefore, lysozyme in blood or intestinal fluid after organ transplantation, particularly small intestine transplantation, is useful as a diagnostic marker for rejection and a medicinal efficacy marker for immunosuppressants.

In the examples of the present invention, the prepared ectopic small intestine transplant model rats were sliced on the 5th, 7th and 10th days after the operation, and the histological rejection determination performed by hematoxylin-eosin (HE) staining was performed. It is a photograph shown. In the Example of this invention, it is a figure which shows the lysozyme density | concentration measured by the protein chip | tip obtained from the ectopic small intestine transplant model rat produced.

Claims (20)

  A diagnostic marker for rejection, characterized by being lysozyme.   The diagnostic marker according to claim 1, wherein the rejection is a rejection occurring after transplantation of the small intestine.   A medicinal efficacy marker for an immunosuppressant characterized by being lysozyme.   4. The drug efficacy marker according to claim 3, wherein the immunosuppressive agent is an immunosuppressive agent administered after small intestine transplantation.   A method for diagnosing the degree of rejection in vertebrates other than humans by measuring the amount of lysozyme in blood or intestinal fluid.   6. The method of claim 5, wherein the individual has been transplanted in the small intestine.   A method for evaluating the efficacy of an immunosuppressive agent by measuring the amount of lysozyme in blood or intestinal fluid in a vertebrate other than a human administered with the immunosuppressive agent.   8. The method of claim 7, wherein the vertebrate is transplanted in the small intestine.   A rejection diagnostic kit comprising an anti-lysozyme antibody.   A drug efficacy evaluation kit for an immunosuppressant containing an anti-lysozyme antibody. A screening method for an effective immunosuppressant in a non-human vertebrate that requires administration of an immunosuppressant,
A screening method comprising using an immunosuppressant drug efficacy marker which is lysozyme. The screening method according to claim 11, comprising:
In each of a plurality of individuals belonging to the vertebrate, before or after administering a different immunosuppressant, blood or intestinal fluid is collected from the individual,
Measuring the lysozyme concentration in the blood or intestinal fluid,
A screening method comprising: comparing the different immunity-suppressing agents before and after administration of the immunity-suppressing agent in lysozyme concentration in blood or intestinal fluid collected from the individual.   The screening method according to claim 12, wherein the individual has been transplanted into the small intestine. In a non-human vertebrate that requires administration of an immunosuppressive agent, a method for determining an appropriate amount of an immunosuppressive agent comprising:
A method comprising using a drug efficacy marker of an immunosuppressant, which is lysozyme. 15. A method according to claim 14, comprising
Collecting blood or intestinal fluid from the vertebrate,
Measuring the lysozyme concentration in the blood or intestinal fluid,
Reduce the lysozyme concentration in the blood or intestinal fluid of the individual to a predetermined expression level using a calibration curve that shows the correlation between the amount of immunosuppressant used and the lysozyme concentration in the blood or intestinal fluid. Determining the dose of the immunosuppressive agent required to effect the treatment.   The method according to claim 14 or 15, wherein the vertebrate is transplanted into the small intestine.   A method for diagnosing the degree of rejection by measuring the amount of at least one of MRP8 and MRP14 in blood or intestinal fluid and the amount of lysozyme in vertebrates other than humans.   In non-human vertebrates administered with an immunosuppressive agent, the efficacy of the immunosuppressive agent is evaluated by measuring the amount of at least one of MRP8 and MRP14 in blood or intestinal fluid and the amount of lysozyme. how to.   A rejection diagnostic kit comprising at least one of an anti-MRP8 antibody and an anti-MRP14 antibody and an anti-lysozyme antibody. A kit for evaluating the efficacy of an immunosuppressant comprising at least one of an anti-MRP8 antibody and an anti-MRP14 antibody and an anti-lysozyme antibody.