WO2018190365A1 - Neuropathic pain marker and use thereof - Google Patents

Abstract

The present invention uses an autoantibody to a small unmyelinated dorsal root ganglion neuron as a neuropathic pain marker.

Description

Neuropathic pain marker and use thereof

The present invention relates to a neuropathic pain marker and use thereof. More specifically, the present invention relates to a neuropathic pain marker, a method for detecting neuropathic pain, and a diagnostic kit for neuropathic pain. This application claims priority based on provisional application No. 62 / 484,406 filed provisionally in the United States on April 12, 2017, the contents of which are incorporated herein by reference.

Neuropathic pain is an intractable disease that includes various pathologies defined as “pain caused by lesions and diseases of the somatosensory nervous system” by the International Association for Pain (IASP). It is one of sexual pain (for example, refer nonpatent literature 1).

Peripheral nerve fibers are classified into Aα fibers, Aβ fibers, Aδ fibers, B fibers, and C fibers according to the presence / absence of myelin sheath, diameter, conduction velocity, and the like. In general, nerve fibers having a larger diameter tend to have a higher conduction speed, and nerve fibers that are myelinated tend to have a higher conduction speed. Among these nerve fibers, Aα fibers, Aβ fibers, Aδ fibers, and B fibers are myelinated fibers, and C fibers are unmyelinated fibers.

Neuropathic pain is thought to be caused mainly by Aδ fiber and C fiber damage. The sharp and sharp pain is borne by Aδ fibers, which are small-diameter myelinated fibers, and the blunt and slow pain is borne by C fibers, which are unmyelinated fibers.

However, the nerve conduction test, which is a general test for peripheral neuropathy, examines Aβ fibers, which are large-diameter myelinated fibers. For this reason, an abnormality cannot be detected even if a peripheral neuropathy test common to patients with neuropathic pain is performed. As a result, many patients with neuropathic pain are determined to be of unknown cause or psychogenic, and have not yet been diagnosed.

Under such background, an object of the present invention is to provide a technique for detecting neuropathic pain.

The present invention includes the following aspects.
[1] Use of autoantibodies against small unmyelinated dorsal root ganglion neurons as a neuropathic pain marker.
[2] The use according to [1], wherein the autoantibody is an anti-Plexin D1 antibody.
[3] A method for detecting neuropathic pain, comprising detecting autoantibodies against small unmyelinated dorsal root ganglion neurons in a blood sample derived from a patient, wherein the autoantibodies are detected. A method showing that the patient is suffering from neuropathic pain.
[4] To detect autoantibodies against small unmyelinated dorsal root ganglion neurons, a human or non-human animal-derived dorsal root ganglion tissue or spinal dorsal horn tissue is contacted with a patient-derived blood sample, Detecting human IgG antibodies bound to ganglion tissue or spinal dorsal horn tissue; detecting myelinated dorsal root ganglion neurons or nerve fibers thereof in the dorsal root ganglion tissue or spinal dorsal horn tissue; Wherein the human IgG antibody binds to a neuron other than the myelinated dorsal root ganglion neuron, indicating that an autoantibody against a small unmyelinated dorsal root ganglion neuron has been detected. [3] the method of.
[5] Detecting a myelinated dorsal root ganglion neuron includes immunostaining the dorsal root ganglion tissue with an anti-S100β antibody, and the neuron immunostained with the anti-S100β antibody is a myelinated dorsal root nerve The method according to [4], which is a nodal neuron.
[6] To detect autoantibodies against small unmyelinated dorsal root ganglion neurons, a human or non-human animal-derived dorsal root ganglion tissue or spinal cord dorsal horn tissue is contacted with a patient-derived blood sample, Detecting a human IgG antibody bound to the ganglion tissue or the dorsal horn tissue of the spinal cord; detecting unmyelinated dorsal root ganglion neurons or nerve fibers thereof in the dorsal root ganglion tissue or the dorsal horn tissue of the spinal cord; The method according to [3], wherein binding of the human IgG antibody to the unmyelinated dorsal root ganglion neuron indicates that an autoantibody against a small unmyelinated dorsal root ganglion neuron has been detected.
[7] Detecting an unmyelinated dorsal root ganglion neuron includes contacting the dorsal root ganglion tissue with isolectin B4, and the neuron to which the isolectin B4 is bound is an unmyelinated dorsal root ganglion neuron. The method according to [6].
[8] The method according to any one of [3] to [7], wherein the human IgG antibody is an IgG2 antibody.
[9] The method according to any one of [3] to [8], wherein the autoantibody is an anti-Plexin D1 antibody.
[10] A method for detecting neuropathic pain, comprising contacting a blood sample derived from a patient with a human cell or non-human animal cell expressing Plexin D1, and detecting a human IgG antibody bound to the cell. When the blood sample is contacted with a cell that does not express Plexin D1, the amount of the human IgG antibody detected is higher than the amount of the human IgG antibody that binds to the cell. A method of showing that it is suffering from neuropathic pain.
[11] A diagnostic kit for neuropathic pain comprising an anti-human IgG antibody and a detection agent for myelinated dorsal root ganglion neurons or a detection agent for unmyelinated dorsal root ganglion neurons.
[12] The diagnostic kit according to [11], wherein the anti-human IgG antibody is an anti-human IgG2 antibody.
[13] The diagnostic kit according to [11] or [12], wherein the drug for detecting myelinated dorsal root ganglion neurons is an anti-S100β antibody.
[14] The diagnostic kit according to any one of [11] to [13], wherein the detection agent for unmyelinated dorsal root ganglion neurons is isolectin B4.
[15] A diagnostic kit for neuropathic pain, comprising an anti-human IgG antibody and a Plexin D1 protein.
[16] The diagnostic kit according to [15], wherein the anti-human IgG antibody is an anti-human IgG2 antibody.

The present invention can provide a technique for detecting neuropathic pain.

(A) to (f) are fluorescence micrographs showing the results of Experimental Example 2. (A) to (d) are fluorescence micrographs showing the results of Experimental Example 2. (A) to (d) are fluorescence micrographs showing the results of Experimental Example 3. (A) to (c) are fluorescence micrographs showing the results of double staining of patient serum and isolectin B4 in Experimental Example 4. (A) to (c) are fluorescence micrographs showing the results of double staining of patient serum and anti-CGRP antibody in Experimental Example 4. (A)-(c) are fluorescence micrographs showing the results of double staining of patient serum and anti-S100β antibody in Experimental Example 4. (A)-(c) are fluorescence micrographs showing the results of double staining of patient serum and anti-TRPV1 antibody in Experimental Example 4. (A) to (c) are fluorescence micrographs showing the results of double staining of patient serum and anti-P2X3 antibody in Experimental Example 4. (A)-(c) are fluorescence micrographs showing the results of double staining of patient serum and anti-CGRP antibody in Experimental Example 5. (A) to (c) are fluorescence micrographs showing the results of double staining of patient serum and isolectin B4 in Experimental Example 5. (A) to (c) are fluorescence micrographs showing the results of double staining of patient serum and anti-PKCγ antibody in Experimental Example 5. (A) is an optical micrograph showing the results of Experimental Example 6. (B) and (c) are fluorescence micrographs showing the results of Experimental Example 6. (A) to (c) are fluorescence micrographs showing the results of double staining of patient serum and anti-TH antibody in Experimental Example 6. (A) to (c) are fluorescence micrographs showing the results of double staining of patient serum and anti-VIP antibody in Experimental Example 6. (A) And (c) is a photograph which shows the result of the western blotting in Experimental example 7. FIG. (B) is a photograph showing the results of SDS-PAGE and silver staining in Experimental Example 7. (A) to (c) are fluorescence micrographs showing the results of Experimental Example 8. (A) is a graph showing the results of quantitative real-time PCR in Experimental Example 9. (B) is a photograph showing the results of Western blotting in Experimental Example 9. (A) to (c) are fluorescence micrographs showing the results of Experimental Example 9. (A) is an optical micrograph showing the results of Experimental Example 9. (B) and (c) are fluorescence micrographs showing the results of Experimental Example 9. (D) is a merged photograph of (a) to (c). (A) And (b) is the fluorescence micrograph which shows the result of Experimental example 10. FIG. (C) is the graph which digitized the result of (a) and (b). (A) to (f) are fluorescence micrographs showing the results of Experimental Example 11. (A) to (d) are photographs showing the results of Western blotting in Experimental Example 12. It is a photograph which shows the result of the western blotting in Experimental example 13.

[Neuropathic pain marker]
In one embodiment, the present invention provides a neuropathic pain marker. As will be described later in the Examples, the inventors have revealed that autoantibodies against small unmyelinated dorsal root ganglion neurons are markers of neuropathic pain. Thus, the detection of autoantibodies against small unmyelinated dorsal root ganglion neurons in the patient serum indicates that the patient has neuropathic pain.

Conventionally, it has been difficult to detect neuropathic pain. On the other hand, neuropathic pain can be easily detected by the marker of this embodiment.

This embodiment can also be said to provide the use of autoantibodies against small unmyelinated dorsal root ganglion neurons as neuropathic pain markers. Alternatively, it can be said to provide a method of using autoantibodies against small unmyelinated dorsal root ganglion neurons as a neuropathic pain marker.

The autoantibody against the small unmyelinated dorsal root ganglion neuron may be an anti-Plexin D1 antibody or an autoantibody against a small unmyelinated dorsal root ganglion neuron other than the anti-Plexin D1 antibody. As will be described later in Examples, the inventors have clarified that anti-Plexin D1 antibody is present in the serum of a patient having neuropathic pain. However, it is presumed that not all autoantibodies to small unmyelinated dorsal root ganglion neurons recognize PlexinD1 alone.

Note that the RefSeqID of the human Plexin D1 protein is NP_055918, and the RefSeqID of the mouse Plexin D1 protein is NP_080652.

The anti-Plexin D1 antibody in a blood sample derived from a patient may be detected by, for example, a lateral flow immunoassay method, an ELISA method, a Western blotting method, or the like. For example, after reacting a blood sample derived from a patient with a solid phase to which Plexin D1 protein is immobilized, a method of detecting an autoantibody bound to Plexin D1 protein with an anti-human IgG antibody, and the like are mentioned.

Plexin D1 is a molecule conventionally known as a neurogenesis guidance factor, a semaphorin receptor involved in immune cell differentiation and activation, and the like. Conventionally, the relationship between Plexin D1 and pain has not been reported.

Detection of the presence of anti-Plexin D1 antibody present in the patient's serum or autoantibodies against unidentified small unmyelinated dorsal root ganglion neurons indicates that the patient has neuropathic pain . That is, by detecting an anti-Plexin D1 antibody present in a patient's serum or an autoantibody against an unidentified small unmyelinated dorsal root ganglion neuron, neuropathic pain that has been conventionally diagnosed as unknown is eliminated. Diagnosis is possible.

The present embodiment is a data collection method for diagnosing whether or not a patient suffers from neuropathic pain, and is an anti-PlexinD1 antibody present in the patient's serum or an unidentified small antigen. It can also be a method comprising detecting the presence of autoantibodies against dorsal root ganglion neurons. The data collection method does not include a step in which a doctor determines the patient's condition.

When an anti-small unmyelinated dorsal root ganglion neuron antibody is detected in the patient's serum, it can be diagnosed as small fiber neuropathy. Small fiber neuropathy can be treated with immunotherapy.

Therefore, by using the presence of autoantibodies against small unmyelinated dorsal root ganglion neurons as an index, it is possible to quickly determine the indication of immunotherapy for the treatment of neuropathic pain, and early diagnosis of neuropathic pain・ Early treatment is possible.

Plexin D1 autoantibody-positive or antigen-unidentified autoantibody-positive neuropathic pain patients with small unmyelinated dorsal root ganglion neurons can be treated differently from patients with neuropathic pain due to other causes It is.

The neuropathic pain in these patients can be treated by immunotherapy such as administration of corticosteroids, administration of immunosuppressants, high-dose immunoglobulin therapy, plasma exchange therapy, and the like. In addition, as a symptomatic treatment, it is effective to administer an analgesic agent such as an SCN9A inhibitor, an SCN10A inhibitor, or a P2X3 receptor antagonist that targets small-diameter unmyelinated fibers.

[Method for detecting neuropathic pain]
(First embodiment)
In one embodiment, the present invention relates to a method for detecting neuropathic pain, comprising detecting an autoantibody against a small unmyelinated dorsal root ganglion neuron in a blood sample derived from a patient, wherein the autoantibody comprises Detecting provides a method for indicating that the patient is suffering from neuropathic pain. Hereinafter, the method of the present embodiment may be referred to as a fluorescent indirect antibody method (IFA).

It can also be said that the method of the first embodiment is a data collection method for diagnosing whether or not a patient suffers from neuropathic pain. The data collection method does not include a step in which a doctor determines the patient's condition.

In the method of the present embodiment, detecting autoantibodies against small unmyelinated dorsal root ganglion neurons involves contacting a patient-derived blood sample with dorsal root ganglion tissue or spinal dorsal horn tissue derived from a human or non-human animal. Detecting a human IgG antibody bound to the dorsal root ganglion tissue or the dorsal horn tissue of the spinal cord; and a myelinated dorsal root ganglion neuron or a nerve fiber thereof in the dorsal root ganglion tissue or the dorsal horn tissue of the spinal cord. The human IgG antibody binds to a neuron other than the myelinated dorsal root ganglion neuron, indicating that an autoantibody against a small unmyelinated dorsal root ganglion neuron has been detected. There may be.

According to the method of the first embodiment, in order to detect an autoantibody that reacts with the dorsal root ganglion tissue or the dorsal horn tissue of the spinal cord, an autoantibody against a small unmyelinated dorsal root ganglion neuron that has not been identified with an antigen is detected. be able to.

The non-human animal is not particularly limited, and examples thereof include mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, monkeys, sheep, pigs, goats, cows, horses and the like.

As will be described later in the Examples, the inventors obtained sera from patients with neuropathic pain, tissue sections of human dorsal root ganglia, tissue sections of human dorsal horn, tissue sections of mouse dorsal root ganglia. It was clarified that autoantibodies against small unmyelinated dorsal root ganglion neurons can be detected by reacting with tissue slices of the dorsal horn of mouse spinal cord and detecting the binding.

Further, detecting the myelinated dorsal root ganglion neuron includes immunostaining the dorsal root ganglion tissue with an anti-S100β antibody, and the neurons immunostained with the anti-S100β antibody It may be determined that it is a neuron. As described later in the Examples, the inventors have revealed that autoantibodies derived from patients with neuropathic pain do not colocalize with anti-S100β antibodies.

In the method of the present embodiment, detecting autoantibodies against small unmyelinated dorsal root ganglion neurons involves contacting a patient-derived blood sample with dorsal root ganglion tissue or spinal dorsal horn tissue derived from a human or non-human animal. Detecting a human IgG antibody bound to the dorsal root ganglion tissue or the spinal dorsal horn tissue, and an unmyelinated dorsal root ganglion neuron or a nerve fiber thereof in the dorsal root ganglion tissue or the spinal dorsal horn tissue. The human IgG antibody bound to the unmyelinated dorsal root ganglion neuron may indicate that an autoantibody against a small unmyelinated dorsal root ganglion neuron has been detected. Good.

Non-human animals are the same as described above. Further, detecting the unmyelinated dorsal root ganglion neuron includes contacting the dorsal root ganglion tissue with isolectin B4, and determining that the neuron to which the isolectin B4 is bound is an unmyelinated dorsal root ganglion neuron. May be. As described later in the Examples, the inventors revealed that autoantibodies derived from patients with neuropathic pain co-localize with isolectin B4.

In the method of the present embodiment, the autoantibody against small unmyelinated dorsal root ganglion neurons contained in a patient-derived blood sample may be an IgG2 antibody. As will be described later in the Examples, the inventors have revealed that the IgG subclass of autoantibodies derived from patients with neuropathic pain is IgG2.

In the method of the present embodiment, the autoantibody against small unmyelinated dorsal root ganglion neurons contained in a patient-derived blood sample may be an anti-Plexin D1 antibody. As described later in Examples, the inventors have clarified that the autoantibody derived from a patient having neuropathic pain is an anti-Plexin D1 antibody.

(Second Embodiment)
In one embodiment, the present invention is a method for detecting neuropathic pain, wherein a human cell or non-human animal cell expressing Plexin D1 is contacted with a blood sample derived from a patient, and the human IgG antibody bound to the cell The amount of the human IgG antibody detected is greater than the amount of the human IgG antibody that binds to the cell when the blood sample is contacted with a cell that does not express Plexin D1. Provides a method of indicating that the patient is suffering from neuropathic pain.

It can also be said that the method of the second embodiment is a data collection method for diagnosing whether or not a patient suffers from neuropathic pain. The data collection method does not include a step in which a doctor determines the patient's condition.

The method of the second embodiment is mainly different from the method of the first embodiment in that the antigen of the autoantibody is limited to Plexin D1 protein.

Non-human animals are the same as described above. The cell that expresses Plexin D1 may be a cell that originally expresses Plexin D1, or may be a cell that expresses Plexin D1 as a result of introducing a Plexin D1 expression vector. For example, as described later in the Examples, HeLa cells can be used as cells that express Plexin D1.

The cell that does not express Plexin D1 may be a cell that originally does not express Plexin D1, may be a cell in which the PLXND1 gene that encodes the Plexin D1 protein is disrupted, or Plexin D1. It may be a cell in which the expression level of is reduced. Specifically, for example, as described later in Examples, HeLa cells into which siRNA for the PLXND1 gene has been introduced can be used as cells that do not express Plexin D1.

In the method of the second embodiment, the autoantibodies contained in the patient-derived blood sample may be IgG2 antibodies.

[Diagnostic kit for neuropathic pain]
(First embodiment)
In one embodiment, the present invention provides a diagnostic kit for neuropathic pain comprising an anti-human IgG antibody and a detection agent for myelinated dorsal root ganglion neurons or a detection agent for unmyelinated dorsal root ganglion neurons. To do.

The kit of this embodiment can be suitably used for the method for detecting neuropathic pain of the first embodiment described above. Specifically, a patient-derived blood sample may be detected by anti-human IgG antibody after contacting a patient-derived blood sample with dorsal root ganglion tissue or spinal dorsal horn tissue derived from a human or non-human animal. The anti-human IgG antibody may be an anti-human IgG2 antibody.

In addition, a detection agent for myelinated dorsal root ganglion neurons or a detection agent for unmyelinated dorsal root ganglion neurons caused the patient-derived IgG antibody to bind to myelinated dorsal root ganglion neurons, or unmyelinated dorsal root ganglion neurons. Can be determined. Anti-S100β antibody can be used as a detection agent for myelinated dorsal root ganglion neurons. Moreover, isolectin B4 is mentioned as a detection agent of an unmyelinated dorsal root ganglion neuron.

If autoantibodies against small unmyelinated dorsal root ganglion neurons are detected, it can be determined that the patient is a patient with neuropathic pain.

(Second Embodiment)
In one embodiment, the present invention provides a diagnostic kit for neuropathic pain comprising an anti-human IgG antibody and a Plexin D1 protein.

According to the kit of this embodiment, it is easily detected whether autoantibodies to Plexin D1 protein are present in a blood sample derived from a patient, for example, by a lateral flow immunoassay method, an ELISA method, a Western blotting method, or the like. be able to.

More specifically, for example, after reacting a blood sample derived from a patient with a solid phase on which Plexin D1 protein is immobilized, autoantibodies bound to Plexin D1 protein are detected with anti-human IgG antibodies, thereby autoantibodies. The presence of can be detected.

In the kit of this embodiment, as the Plexin D1 protein, the full length of the Plexin D1 protein may be used, the extracellular domain of the Plexin D1 protein may be used, or the Plexin D1 protein effective for detecting autoantibodies. An epitope part is specified, and a shorter partial peptide may be used. As the Plexin D1 protein, for example, a recombinant human Plexin D1 protein (model “4160-PD”, R & D Systems, the amino acid sequence is shown in SEQ ID NO: 8) described later in Examples can be used.

When an autoantibody against Plexin D1 protein is detected, it can be determined that the patient is a neuropathic pain patient. The anti-human IgG antibody may be an anti-human IgG2 antibody.

[Other Embodiments]
In one embodiment, the present invention is a method for diagnosing and treating neuropathic pain in a patient, comprising collecting a blood sample from the patient, and a small unmyelinated dorsal root ganglion neuron in the blood sample. Detecting whether there is an autoantibody against, and diagnosing that the patient is neuropathic pain and having been diagnosed with neuropathic pain when the autoantibody is present In addition, the patient is administered an effective amount of a drug selected from the group consisting of corticosteroids, immunosuppressants, SCN9A inhibitors, SCN10A inhibitors, P2X3 receptor antagonists, or massive immunoglobulin therapy Alternatively, a method is provided comprising performing plasma exchange therapy.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[Experimental Example 1]
(Collection of blood sample)
Blood samples were collected from patients with neuropathic pain. For patients with neuropathic pain, the diagnostic criteria for neuropathic pain proposed by the International Pain Society (Finnerup NB, et al., Neuropathic pain: an updated grading system for research and clinical practice., Pain, vol. 157 (8), 1599-1606, 2016.), 110 patients who met the probable and define criteria were included.

The breakdown of patients with neuropathic pain is 22 patients with atopic myelitis, 17 patients with optic neuromyelitis (NMOSD), 15 patients with relapsing-remitting multiple sclerosis (RRMS), multiple chronic inflammatory demyelinating 14 patients with neuritis (CIDP), 10 patients with Sjogren's syndrome with spinal root peripheral neuritis, 10 patients with neurosarcoidosis, 6 patients with Churg Strauss, patients with systemic lupus erythematosus (SLE) with myelitis and peripheral neuritis 4 patients, 3 patients with neuro-Behcet's disease (nBD), 2 patients with acromegaly, 2 patients with drug-induced neuropathy, 2 patients with vitamin deficiency neuropathy, 1 patient with neuropathy positive for anti-SGPG antibody, Guillain Valley There were 1 syndrome patient and 1 cryoglobulinemia patient.

As controls, blood samples were also collected from healthy individuals and patients without neuropathic pain (total of 50 patients). The breakdown of 50 is 20 healthy individuals, 20 patients with neurodegenerative diseases (of which 6 are amyotrophic lateral sclerosis, 4 are multisystem atrophy, and 3 are spinocerebellar degeneration) 2 had Parkinson's disease, 2 had normal pressure hydrocephalus, 1 had Alzheimer's disease, 1 had dementia, and 1 had basal ganglia degeneration. ), 10 patients with collagen vascular disease (of which 4 were systemic lupus erythematosus, 4 were Behcet's disease, and 2 were Sjogren's syndrome).

[Experiment 2]
(Detection of autoantibodies against dorsal root ganglion neurons)
Autoantibodies against dorsal root ganglion neurons in blood samples collected in Experimental Example 1 using tissue sections of mouse dorsal root ganglion (DRG) and mouse spinal cord (spinal cord, SC) Was detected.

<Preparation of tissue section>
8-10 week old male C57BL / 6 mice were anesthetized. Subsequently, it was perfused with phosphate buffered saline (PBS). Subsequently, perfusion fixation was performed with 4% paraformaldehyde. Subsequently, the spinal cord and dorsal root ganglia at the lumbar spinal level (L4-L6) were removed, and each tissue was fixed with 10% formaldehyde. Subsequently, each tissue was embedded in paraffin, a 4 μm-thick tissue section was prepared, and attached to a glass slide.

<Reaction with blood sample>
The prepared tissue section was immersed in xylene for 5 minutes three times to deparaffinize. Subsequently, immersing the tissue section in 100% ethanol for 5 minutes was repeated twice and hydrated. Subsequently, antigen activation treatment was performed. Specifically, the tissue sections were boiled in citrate buffer (pH 6.0) at 120 ° C. for 10 minutes, slowly cooled at room temperature over 15 minutes, and washed twice with PBS for 5 minutes.

Subsequently, 10% goat serum / PBS was brought into contact with the tissue section and allowed to stand at room temperature for 30 minutes for blocking. Subsequently, a 60-fold diluted blood sample (serum) as a primary antibody was brought into contact with the tissue section and left at 37 ° C. for 1 hour to carry out an antigen-antibody reaction. Subsequently, the plate was washed twice with PBS for 5 minutes.

Subsequently, a secondary antibody (Alexa 488-labeled anti-human IgG antibody, Thermo Fisher Scientific) diluted 500-fold was brought into contact with the tissue section and allowed to stand at room temperature for 1 hour to carry out an antigen-antibody reaction. Subsequently, the plate was washed twice with PBS for 5 minutes.

Subsequently, the tissue section was sealed and observed with a confocal laser microscope (model “LSM510”, Carl Zeiss). As a result, it was clarified that the serum derived from healthy subjects hardly contains autoantibodies that react with tissue sections of mouse dorsal root ganglia and mouse spinal cord.

On the other hand, sera from 110 patients out of 110 patients with neuropathic pain are present in small unmyelinated dorsal root ganglion neurons and spinal dorsal horns of tissue sections of mouse dorsal root ganglia It was revealed that it contained autoantibodies that responded to its nerve endings.

FIG. 1 (a) is a representative fluorescence micrograph showing the result of a tissue section of a mouse dorsal root ganglion reacted with a control serum. The scale bar indicates 50 μm. 1 (b)-(f) show autoantibodies detected in tissue sections of mouse dorsal root ganglia reacted with serum from patients with neuropathic pain (cases 1-5, respectively). It is the fluorescence micrograph which shows the typical result done. The scale bar indicates 50 μm.

FIG. 2 (a) is a representative fluorescence micrograph showing the result of a tissue section of a mouse spinal cord reacted with serum from the same healthy person as in FIG. 1 (a). The scale bar indicates 50 μm. FIG. 2 (b) is a fluorescence micrograph of the same sample as FIG. 2 (a) taken at high magnification. The scale bar indicates 50 μm.

FIG. 2 (c) is a representative fluorescence micrograph showing the result of a tissue section of a mouse spinal cord reacted with serum from the same patient (case 5) as in FIG. 1 (f). The scale bar indicates 50 μm. FIG. 2 (d) is a fluorescence micrograph of the same sample as FIG. 2 (c) taken at high magnification. The scale bar indicates 50 μm.

Hereinafter, the method of this experimental example may be referred to as a fluorescent indirect antibody method (tissue-based indirect immunofluorescence assay, IFA).

The results are summarized in Table 1 below. In Table 1, “NeP” represents neuropathic pain, “NS” represents no significant difference, and “IFA” represents the fluorescent indirect antibody method. Mann-Whitney U test was used for comparison of continuous variables. In addition, chi-square test was used for comparison of variables between categories between patients with and without NeP, and Fisher exact test was used when chi-square test could not be applied.

In addition, Table 2 below shows the clinical characteristics of 11 patients who contained autoantibodies reactive to tissue sections of mouse dorsal root ganglia.

[Experiment 3]
(Examination of subclasses of autoantibodies against dorsal root ganglion neurons)
The IgG subclass of autoantibodies was examined for sera from 11 patients that contained autoantibodies reactive to tissue sections of mouse dorsal root ganglia detected in Experimental Example 2.

Specifically, in the secondary antibody reaction, experimental examples except that FITC-labeled mouse monoclonal antibodies (Sigma Aldrich) specific for human IgG1, IgG2, IgG3, and IgG4 were respectively diluted 50 times and reacted. The same fluorescent indirect antibody method as 2 was performed.

3 (a) to 3 (d) are representative fluorescence micrographs showing typical results of detecting autoantibodies of IgG1, IgG2, IgG3, and IgG4, respectively. The scale bar indicates 50 μm. As a result, it was revealed that the IgG subclass of autoantibodies was IgG2 in all 11 patient-derived sera.

[Experimental Example 4]
(Examination of subtypes of dorsal root ganglion neurons to which autoantibodies respond)
About the autoantibodies derived from 11 patients reacting to the tissue section of the mouse dorsal root ganglion detected in Experimental Example 2, small unmyelinated dorsal root ganglion neurons (unmyelinated C fiber type dorsal root ganglion neurons) Whether or not to selectively bind was examined.

<< Double staining with isolectin B4 >>
In the primary antibody reaction, in addition to patient serum diluted 60-fold, isolectin B4 (Iselectin GS-IB ₄ From Griffinia simplicifolia, Alexa Fluor 594 Conjugate, thermofischer science, a marker of non-peptide C fiber type dorsal root ganglion neurons The fluorescent indirect antibody method was carried out in the same manner as in Experimental Example 2 except that the reaction was carried out after diluting 500 times.

FIG. 4 (a) is a representative fluorescence micrograph showing the result of a tissue section of a mouse dorsal root ganglion reacted with serum from a patient with neuropathic pain. FIG. 4 (b) is a representative fluorescence micrograph showing the result of detecting the binding of isolectin B4 (IB4) in the same field of view as FIG. 4 (a). FIG. 4C is a merged photograph of FIGS. 4A and 4B. The scale bar indicates 50 μm. As a result, it was revealed that patient-derived autoantibodies colocalized with isolectin B4.

《Double staining with anti-CGRP antibody》
In the primary antibody reaction, in addition to patient serum diluted 60-fold, an antibody against a calcitonin gene-related peptide (CGRP) that is a marker of peptidic C-fiber dorsal root ganglion neurons (rabbit polyclonal antibody, Yauchihara) The indirect fluorescent antibody method was performed in the same manner as in Experimental Example 2 except that the laboratory was diluted 500 times and reacted.

FIG. 5 (a) is a representative fluorescence micrograph showing the result of a tissue section of a mouse dorsal root ganglion reacted with serum derived from a patient with neuropathic pain. FIG. 5 (b) is a representative fluorescence micrograph showing the result of detecting the binding of anti-CGRP antibody in the same field of view as FIG. 5 (a). FIG. 5C is a merged photograph of FIGS. 5A and 5B. The scale bar indicates 50 μm. As a result, it was revealed that patient-derived autoantibodies colocalized only partly with CGRP.

<< Double staining with anti-S100β antibody >>
In the primary antibody reaction, in addition to patient serum diluted 60 times, the myelinated nerve fibers, Aβ fiber type dorsal root ganglion neurons and Aδ fiber type dorsal root ganglion neurons, and S100β which is a marker of satellite glial cells A fluorescent indirect antibody method was performed in the same manner as in Experimental Example 2 except that the antibody (rabbit polyclonal antibody, Abcam) was diluted 500 times and reacted.

FIG. 6 (a) is a representative fluorescence micrograph showing the result of a tissue section of a mouse dorsal root ganglion reacted with serum derived from a patient with neuropathic pain. FIG. 6 (b) is a representative fluorescence micrograph showing the result of detecting the binding of anti-S100β antibody in the same field of view as FIG. 6 (a). FIG. 6C is a merged photograph of FIGS. 6A and 6B. The scale bar indicates 50 μm. As a result, it was revealed that patient-derived autoantibodies do not colocalize with S100β.

<< Double staining with anti-TRPV1 antibody >>
From the above results, it was revealed that autoantibodies derived from patients with neuropathic pain specifically bind to small unmyelinated dorsal root ganglion neurons.

Therefore, in the primary antibody reaction, in addition to patient serum diluted 60 times, an antibody (rabbit polyclonal antibody) against transient receptor potential vanilloid 1 (TRPV1), which is known to be involved in pain perception, was diluted 1,000 times. Then, the fluorescent indirect antibody method was performed in the same manner as in Experimental Example 2 except that the reaction was performed.

FIG. 7 (a) is a representative fluorescence micrograph showing the result of a tissue section of a mouse dorsal root ganglion reacted with serum derived from a patient with neuropathic pain. FIG. 7 (b) is a representative fluorescence micrograph showing the result of detecting the binding of anti-TRPV1 antibody in the same field of view as FIG. 7 (a). FIG. 7C is a merged photograph of FIGS. 7A and 7B. The scale bar indicates 50 μm. As a result, it was revealed that some of the patient-derived autoantibodies reacted with TRPV1-positive dorsal root ganglion neurons. This result further supports that patient-derived autoantibodies are associated with neuropathic pain.

<< Double staining with anti-P2X3 antibody >>
In the primary antibody reaction, in addition to patient serum diluted 60 times, antibody (rabbit polyclonal antibody, Abcam) against P2X purinoceptor 3 (P2X3), which is known to be involved in pain perception, was diluted 500 times and reacted. The fluorescent indirect antibody method was performed in the same manner as in Experimental Example 2 except for the points described above.

FIG. 8 (a) is a representative fluorescence micrograph showing the result of a tissue section of a mouse dorsal root ganglion reacted with serum derived from a patient with neuropathic pain. FIG. 8 (b) is a representative fluorescence micrograph showing the result of detecting the binding of anti-P2X3 antibody in the same field of view as FIG. 8 (a). FIG. 8C is a merged photograph of FIGS. 8A and 8B. The scale bar indicates 50 μm. As a result, it was revealed that patient-derived autoantibodies mainly reacted with P2X3-positive dorsal root ganglion neurons. This result further supports that patient-derived autoantibodies are associated with neuropathic pain.

[Experimental Example 5]
(Examination of autoantibody reactivity to dorsal spinal cord tissue sections)
Regarding the autoantibodies derived from 11 patients reacting with the tissue section of the mouse dorsal root ganglion detected in Experimental Example 2, the reactivity of the mouse dorsal spinal cord with the tissue section was examined.

《Double staining with anti-CGRP antibody》
In the primary antibody reaction, in addition to patient serum diluted 60-fold, an antibody against a calcitonin gene-related peptide (CGRP) that is a marker of peptidic C-fiber dorsal root ganglion neurons (rabbit polyclonal antibody, Yauchihara) The indirect fluorescent antibody method was performed in the same manner as in Experimental Example 2 except that the laboratory was diluted 500 times and reacted.

FIG. 9 (a) is a representative fluorescence micrograph showing the result of a tissue section of the mouse dorsal spinal cord reacted with serum from a patient with neuropathic pain. FIG. 4B is a representative fluorescence micrograph showing the result of detecting the binding of the anti-CGRP antibody in the same field of view as FIG. FIG. 4C is a merged photograph of FIGS. 4A and 4B. The scale bar indicates 50 μm. As a result, it was revealed that some of the patient-derived autoantibodies reacted with CGRP-positive axon terminals located in the dorsal horn layer I and IIo layers of the spinal cord.

<< Double staining with isolectin B4 >>
In the primary antibody reaction, in addition to patient serum diluted 60-fold, isolectin B4 (Iselectin GS-IB ₄ From Griffinia simplicifolia, Alexa Fluor 594 Conjugate, thermofischer science, a marker of non-peptide C fiber type dorsal root ganglion neurons The fluorescent indirect antibody method was carried out in the same manner as in Experimental Example 2 except that the reaction was carried out after diluting 500 times.

FIG. 10 (a) is a representative fluorescence micrograph showing the result of a tissue section of a mouse dorsal spinal cord reacted with serum from a patient having neuropathic pain. FIG. 10 (b) is a representative fluorescence micrograph showing the result of detecting the binding of isolectin B4 in the same field of view as FIG. 10 (a). FIG. 10C is a merged photograph of FIGS. 10A and 10B. The scale bar indicates 50 μm. As a result, it was revealed that many of the autoantibodies derived from patients reacted with isolectin B4 staining axon terminals located in the spinal cord dorsal horn IIi layer.

《Double staining with anti-PKCγ antibody》
In the primary antibody reaction, in addition to the patient serum diluted 60 times, the fluorescent indirect antibody method similar to Experimental Example 2 was used except that the anti-PKCγ antibody (rabbit polyclonal antibody, Santa Cruz) was diluted 500 times and reacted. went.

FIG. 11 (a) is a representative fluorescence micrograph showing the result of a tissue section of the mouse dorsal spinal cord reacted with serum from a patient with neuropathic pain. FIG. 11 (b) is a representative fluorescence micrograph showing the result of detecting the binding of anti-PKCγ antibody in the same field of view as FIG. 11 (a). FIG. 11C is a merged photograph of FIGS. 11A and 11B. The scale bar indicates 50 μm.

As a result, the majority of patient-derived autoantibodies did not overlap with the PKCγ positive bands located in the dorsal horn IIi layer and the dorsal horn III layer on the abdominal side, and reacted more to the dorsal side. It became clear.

The above results indicate that the binding of patient-derived autoantibodies is restricted to the dorsal horn layer I and IIo of the spinal cord where the axonal terminal of the C-fiber afferent nerve is located.

[Experimental Example 6]
(Examination of reactivity of autoantibodies to post-node autonomic nerve C fibers)
The reactivity of the autoantibodies derived from 11 patients reacting with the tissue section of the mouse dorsal root ganglion detected in Experimental Example 2 was examined for the reactivity to the autonomic nerve C fibers after the node.

The autonomic nerve C fiber is present in the dermis (inner layer of the skin) and shows a distribution pattern similar to protein gene product 9.5 (PGP 9.5), which is a general nerve fiber marker.

First, the binding pattern in the skin of the hind foot soles of patient-derived autoantibodies was examined. FIG. 12 (a) is an optical micrograph showing the result of hematoxylin-eosin staining of a tissue section of the skin of the hind foot sole of a mouse. The scale bar indicates 50 μm. In FIG. 12A, “dermis” indicates the dermis and “epidermis” indicates the epidermis.

FIG. 12 (b) is a representative fluorescence micrograph showing the result of immunostaining of the control serum. In the primary antibody reaction, the fluorescent indirect antibody method similar to Experimental Example 2 except that the anti-PGP9.5 antibody (rabbit polyclonal antibody, Abcam) was diluted 500-fold in addition to the control serum diluted 60-fold. Went. In addition, nuclei were stained with 4 ', 6-diamidino-2-phenylindole (DAPI). The scale bar indicates 50 μm. An enlarged photograph of the arrow is shown in the lower left frame. As a result, the control serum was not reactive with mouse skin.

FIG. 12 (c) is a representative fluorescence micrograph showing the results of immunostaining of patient-derived autoantibodies. In the primary antibody reaction, the fluorescent indirect antibody method similar to Experimental Example 2 except that the anti-PGP9.5 antibody (rabbit polyclonal antibody, Abcam) was diluted 500-fold in addition to the patient serum diluted 60-fold. Went. Also, nuclei were stained with DAPI. The scale bar indicates 50 μm. An enlarged photograph of the arrow is shown in the lower left frame. As a result, it was revealed that patient-derived autoantibodies bound to the epidermis and PGP9.5 positive skin nerve fibers.

《Double staining with anti-TH antibody》
Subsequently, in order to identify the autonomic nerve fiber to which the autoantibody derived from the patient binds to the tissue section of the skin of the hind foot sole of the mouse, the antibody against tyrosine hydroxylase (TH), which is a sympathetic nerve marker, is used. Double staining was performed.

In the primary antibody reaction, the fluorescent indirect antibody method was performed in the same manner as in Experimental Example 2 except that the anti-TH antibody (rabbit polyclonal antibody, Abcam) was diluted 500 times and reacted in addition to patient serum diluted 60 times. It was. Also, nuclei were stained with DAPI.

FIG. 13 (a) is a representative fluorescence micrograph showing the result of the tissue section of the skin of the hind paw plantar of the mouse reacted with serum derived from a patient with neuropathic pain. FIG. 13 (b) is a representative fluorescence micrograph showing the result of detecting anti-TH antibody binding in the same field of view as FIG. 13 (a). FIG. 13C is a merged photograph of FIGS. 13A and 13B. The scale bar indicates 50 μm. As a result, it was revealed that patient-derived autoantibodies do not stain TH-positive nerve fibers.

<< Double staining with anti-VIP antibody >>
Subsequently, double staining with an antibody against vasoactive intestinal peptide (VIP), which is a parasympathetic nerve marker, was performed on a tissue section of the skin of the hind foot sole of the mouse.

In the primary antibody reaction, the fluorescent indirect antibody method similar to Experimental Example 2 was used except that the anti-VIP antibody (rabbit polyclonal antibody, Immunostar) was diluted 500 times and reacted in addition to 60-fold diluted patient serum. went. Also, nuclei were stained with DAPI.

FIG. 14 (a) is a representative fluorescence micrograph showing the result of a tissue section of the skin of the hind paw foot of a mouse reacted with serum derived from a patient with neuropathic pain. FIG. 14 (b) is a representative fluorescence micrograph showing the result of detecting the binding of anti-VIP antibody in the same field of view as FIG. 14 (a). FIG. 14C is a merged photograph of FIGS. 14A and 14B. The scale bar indicates 50 μm. As a result, it was revealed that patient-derived autoantibodies colocalized with VIP-positive nerve fibers.

From the above results, it has been clarified that autoantibodies derived from patients with neuropathic pain also react to parasympathetic nerve C fibers, which are post-node autonomic nerves.

[Experimental Example 7]
(Identification of proteins to which autoantibodies react)
Lumbar dorsal root ganglia of 10-12 week old male C57BL / 6 mice were collected and immediately frozen in liquid nitrogen. Subsequently, the frozen tissue was homogenized in RIPA buffer (Nacalai Tesque) supplemented with Triton X-100 to a final concentration of 1%. Subsequently, the supernatant was collected by centrifugation at 10,000 × g for 30 minutes at 4 ° C. to obtain a mouse dorsal root ganglion-derived protein extract.

Subsequently, the mouse dorsal root ganglion-derived protein extract was subjected to Western blotting, and as a result of detection with a patient-derived autoantibody that reacted with tissue sections of the mouse dorsal root ganglion, 10 patients out of 11 patients A band having a common immunoreactivity with the antibody was detected. The molecular weight of the band was about 220 kDa. On the other hand, as a result of detection with serum derived from a patient not having neuropathic pain and serum derived from a healthy person, no band having immunoreactivity was detected.

FIG. 15 (a) is a photograph showing the result of Western blotting. In FIG. 15 (a), “IFA positive” indicates that the result of the fluorescent indirect antibody method is positive, “IFA negative” indicates that the result of the fluorescent indirect antibody method is negative, and “NeP Pt.1”. 2, 5, 6, 9, 11 "represent patients 1, 2, 5, 6, 9, 11 with neuropathic pain, respectively, and" HC "represents a healthy person. Moreover, the band enclosed with the line is a band detected in common by 10 patient-derived autoantibodies out of 11 patients.

<< Immunoprecipitation, polyacrylamide gel electrophoresis and silver staining >>
From the patient sera, four IgG subclasses of IgG were purified using Protein G HP SpinTrap (GE Healthcare Bioscience).

Also, 300 μL of mouse dorsal root ganglion-derived protein extract diluted to 1 mg / mL and 0.1 mg of FG beads-Protein G beads (Tamakawa Seiki) were incubated at 4 ° C. for 15 minutes. Subsequently, magnetic separation was performed to remove non-specific protein G adsorbate in the protein extract and crude purification was performed.

Subsequently, 3 μg of serum IgG was added to 300 μL of the crudely purified protein extract and incubated at 4 ° C. for 1 hour to cause an antigen-antibody reaction.

Subsequently, 240 μL of the above protein-serum IgG reaction solution was added to 0.1 mg of FG beads-Protein G beads (Tamakawa Seiki) and dispersed. Subsequently, the mixture was incubated at 4 ° C. for 2 hours while stirring with a rotator to perform a binding reaction between the beads and IgG.

Subsequently, after performing magnetic separation and removing the supernatant, 200 μL of 150 mM KCl buffer (150 mM KCl, 20 mM HEPES-NaOH, 1 mM MgCl ₂ , 0.2 mM CaCl ₂ , 0.2 mM EDTA, 10% (v / v The beads were washed a total of 3 times with glycerol, 0.1% NP-40, 0.2 mM PMSF).

Subsequently, 40 μL of 1 × Dye (4 × Dye: 0.25 M Tris-HCl, 8% SDS, 40% glycerol, 20% 2-mercaptoethanol, 0.002% BPB) was added and dispersed, and then 98 Incubated at 5 ° C. for 5 minutes.

Subsequently, magnetic separation was performed at room temperature, and the supernatant was collected to obtain a sample containing the target protein. Subsequently, the autoantigen protein sample obtained by immunoprecipitation was subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE), silver staining was performed to confirm and cut out the band containing the autoantigen protein, and LC-MS / MS Analysis was performed.

FIG. 15 (b) is a photograph showing the results of SDS-PAGE and silver staining. In FIG. 15 (b), lane 1 is a molecular weight marker, lane 2 is a mouse dorsal root ganglion-derived protein extract, lane 3 is a negative control immunoprecipitation sample, and lane 4 contains an autoantigen protein. Immunoprecipitation sample. The arrow indicates a band of autoantigen protein slightly larger than 220 kDa.

FIG. 15 (c) is a photograph showing the result of subjecting the same sample as in FIG. 15 (b) to Western blotting and detecting with a patient-derived autoantibody. In FIG. 15 (c), lane 1 is a molecular weight marker, lane 2 is a mouse dorsal root ganglion-derived protein extract, lane 3 is a negative control immunoprecipitation sample, and lane 4 contains an autoantigen protein. Immunoprecipitation sample. The arrow indicates a band of autoantigen protein slightly larger than 220 kDa.

Subsequently, the analysis results by LC-MS / MS were analyzed with analysis software, and autoantigen candidate proteins were identified. As a result, a peptide fragment having the amino acid sequence of “RTITVAGERF” (SEQ ID NO: 1) was detected. This peptide fragment coincided with the 1087th to 1096th amino acids of the amino acid sequence of plexin D1 protein.

Plexin D1 has a theoretical molecular weight of approximately 212 kDa. This almost coincided with the molecular weight of the band detected by Western blotting. Plexin D1 is one of the largest molecular weight glycoproteins in nerve tissue. However, the expression of Plexin D1 in human dorsal root ganglia and spinal cord has not been reported.

[Experimental Example 8]
(Examination of Plexin D1 expression in human dorsal root ganglia and spinal cord tissue sections)
Tissue sections of human lumbar dorsal root ganglia and spinal cord from dead donors were prepared. Subsequently, each tissue section was immunostained and observed with a fluorescence microscope (model “BZ-X700”, Keyence Corporation).

FIG. 16 (a) is a fluorescence micrograph showing the result of staining a tissue section of a human dorsal root ganglion with an anti-human Plexin D1 antibody (goat polyclonal antibody, R & D Systems).

FIG. 16B shows a neurofilament heavy chain (marker of Aβ fiber-type dorsal root ganglion neurons and Aδ fiber-type dorsal root ganglion neurons, which are myelinated fibers, in the same field of view as FIG. 16A. The results of staining with an antibody against NFH (anti-human phosphorylated NFH antibody, model “SMI31”, mouse monoclonal antibody, Covance; and anti-human non-phosphorylated NFH antibody, model “SMI32”, mouse monoclonal antibody, Covance) It is a fluorescence micrograph shown. Also, nuclei were stained with DAPI.

FIG. 16 (c) is a merged photograph of FIG. 16 (a) and FIG. 16 (b). The scale bar indicates 50 μm.

FIG. 16 (d) is a fluorescence micrograph showing the result of staining a tissue section of human spinal cord with anti-human Plexin D1 antibody (goat polyclonal antibody, R & D Systems).

FIG. 16 (e) shows an anti-human NFH antibody (anti-human phosphorylated NFH antibody, model “SMI31”, mouse monoclonal antibody, Covance; and anti-human non-phosphorylated NFH antibody in the same field of view as FIG. 16 (d). , Model “SMI32”, mouse monoclonal antibody, Covance)). Also, nuclei were stained with DAPI.

FIG. 16 (f) is a merge of the photos of FIG. 16 (d) and FIG. 16 (e). The scale bar indicates 100 μm.

As a result, it was revealed that Plexin D1 does not co-localize with NFH-positive myelinated dorsal root ganglion neurons. On the dorsal side of the spinal cord, NFH is mainly present in the dorsal column (PC) and the dorsal horn deep layer (DDH; spinal dorsal horn III to V layers), and more in the dorsal horn superficial layer (SDH). There were few.

The above results indicate that Plexin D1 is present in the unmyelinated dorsal root ganglion neurons and their axon terminals located in the dorsal horn superficial layer, and this localization pattern is the binding pattern of patient-derived autoantibodies. It was similar.

[Experimental Example 9]
(Examination of reactivity of autoantibodies derived from patients to Plexin D1)
<Suppression of PLXND1 gene expression by siRNA>
HeLa cells (human cervical cancer-derived cell line) expressing Plexin D1, siRNA against PLXND1 gene (model “s23094”, Thermo Fisher Scientific) and control siRNA (model), which are genes encoding Plexin D1 protein "# 4390843", Thermo Fisher Scientific) was introduced, and the mRNA expression level of the PLXND1 gene was quantified by quantitative real-time PCR. In addition, the expression level of Plexin D1 protein was examined at the protein level by Western blotting.

In quantitative real-time PCR, mRNA of the KIF11 gene was amplified as a positive control. In addition, GAPDH gene mRNA was amplified as a reference gene. For quantitative real-time PCR, Fast SYBR Green Master Mix (Thermo Fisher Scientific) was used, and PCR was performed with a StepOnePlus real-time PCR system (Thermo Fisher Scientific).

As PLXND1-specific primers, PLXND1 Fwd (5′-AATGGGCGGAACATCGTCAAG-3 ′, SEQ ID NO: 2) and PLXND1 Rev (5′-CGAGACTGGTTGGAAACACAG-3 ′, SEQ ID NO: 3) were used. Further, KIF11 Fwd (5′-TGTTTGATGATCCCCGTAACAAG-3 ′, SEQ ID NO: 4) and KIF11 Rev (5′-CTGAGTGGGAACGACTAGAGT-3 ′, SEQ ID NO: 5) were used as KIF11 specific primers. Further, GAPDH Fwd (5′-ACCCACTCCTCCACCTTTGAC-3 ′, SEQ ID NO: 6) and GAPDH Rev (5′-TGTTGCTGTAGCCAAATTCGTT-3 ′, SEQ ID NO: 7) were used as GAPDH specific primers.

FIG. 17 (a) is a graph showing the results of quantitative real-time PCR. In FIG. 17 (a), “Scrambled siRNA” indicates the result of introducing the control siRNA, and “PLXND1 siRNA” indicates the result of introducing the siRNA for the PLXND1 gene. As a result, it was confirmed that by introducing siRNA into the PLXND1 gene, the mRNA expression level of the PLXND1 gene could be reduced by an average of 87%.

FIG. 17 (b) is a photograph showing the result of examining the expression of Plexin D1 protein by Western blotting. Β-actin protein was detected as a loading control.

In FIG. 17B, “Scramble siRNA” indicates the result of introducing the control siRNA, and “PLXND1 siRNA” indicates the result of introducing the siRNA for the PLXND1 gene. As a result, it was confirmed that the expression level of Plexin D1 protein could be reduced by 82% by introducing siRNA into the PLXND1 gene.

<Examination of autoantibody reactivity>
An siRNA against the PLXND1 gene was introduced into HeLa cells and allowed to react with autoantibodies from patients with neuropathic pain. For comparison, HeLa cells into which siRNA was not introduced were also prepared.

First, BioCoat Collagen I culture slide 8 well (Corning) was added with a preparation solution containing 7.5 nM final concentration of siRNA and 0.45 μL of Lipofectamine RNAiMAX (Invitrogen) per well to cover the whole well surface, 15 Let stand at room temperature for minutes.

Subsequently, HeLa cells were seeded at 8 × 10 ³ cells / well, cultured under conditions of 37 ° C. and 5% CO ₂ , and reverse transfection was performed. Subsequently, the medium was changed after 48 hours. Subsequently, 96 hours after reverse transfection, the medium was removed, 4% paraformaldehyde / phosphate buffer (Wako Pure Chemical Industries) was added, and the mixture was allowed to stand at room temperature for 10 minutes, and then the cells were washed with ice-cold PBS (−). Washed 3 times.

Subsequently, PBS (-) containing 0.1% Tween 20 (Sigma-Aldrich) was added to each well, left at room temperature for 10 minutes to perform membrane permeation treatment, and then washed twice with PBS (-). . Subsequently, PBS (-) containing 1% gelatin was added to each well and allowed to stand at room temperature for 1 hour for blocking.

Subsequently, patient-derived serum IgG diluted 1,000-fold in PBS (-) containing 1% bovine serum albumin (BSA), and anti-Plexin D1 antibody diluted 200-fold (goat polyclonal antibody, R & D Systems) A primary antibody reaction solution containing the solution was added, and the mixture was allowed to stand at room temperature for 1 hour to carry out a primary antibody reaction, and then washed three times with PBS (−). For comparison, a sample prepared by reacting control serum IgG instead of patient-derived serum IgG was also prepared.

Subsequently, Alexa Fluor 488-labeled goat anti-human IgG (H + L) antibody (Thermo Fisher Scientific) diluted 1,000 times, Alexa Fluor 594-labeled rabbit anti-goat IgG (H + L) antibody (Thermo) diluted 1,000 times. After adding a secondary antibody reaction solution containing Fischer Scientific) and 0.1 μg / mL DAPI, the mixture was allowed to stand at room temperature for 1 hour under light shielding, followed by a secondary antibody reaction, and then PBS (−) And washed 3 times. Subsequently, it was observed with a fluorescence microscope (model “BZ-X710”, Keyence Corporation).

FIG. 18 (a) is a representative fluorescence micrograph showing the result of detection of control IgG bound to HeLa cells into which no siRNA has been introduced. FIG. 18 (b) is a fluorescence micrograph showing the result of detecting the binding of anti-Plexin D1 antibody in the same field of view as FIG. 18 (a). FIG. 18C is a merged photograph of FIGS. 18A and 18B. The scale bar indicates 100 μm.

FIG. 18 (d) is a representative fluorescence micrograph showing the result of detecting patient-derived autoantibodies bound to HeLa cells into which no siRNA has been introduced. FIG. 18 (e) is a fluorescence micrograph showing the result of detecting the binding of anti-Plexin D1 antibody in the same field of view as FIG. 18 (d). FIG. 18F is a merged photograph of FIGS. 18D and 18E. The scale bar indicates 100 μm.

As a result, it was revealed that patient-derived autoantibodies bound to HeLa cells, whereas control IgG did not bind to HeLa cells. It was also revealed that patient-derived autoantibodies colocalized with anti-Plexin D1 antibody.

FIG. 19 (a) is a photomicrograph of HeLa cells into which control siRNA has been introduced, observed in a bright field. FIG. 19B is a fluorescence micrograph showing the result of detecting the binding of a patient-derived autoantibody (Case 5) in the same field of view as FIG. FIG. 19 (c) is a fluorescence micrograph showing the result of detecting the binding of anti-Plexin D1 antibody in the same field of view as FIG. 19 (a). FIG. 19D is a merged photograph of FIGS. 19A, 19B, and 19C. The scale bar indicates 25 μm.

FIG. 19 (e) is a photomicrograph of bright field observation of HeLa cells into which siRNA for the PLXND1 gene has been introduced. FIG. 19 (f) is a fluorescence micrograph showing the result of detecting the binding of the patient-derived autoantibody (Case 5) in the same field of view as FIG. 19 (e). FIG. 19 (g) is a fluorescence micrograph showing the result of detecting the binding of anti-Plexin D1 antibody in the same field of view as FIG. 19 (e). FIG. 19 (h) is a merge of the photos of FIG. 19 (e), FIG. 19 (f) and FIG. 19 (g). The scale bar indicates 25 μm.

As a result, it was revealed that the introduction of siRNA against the PLXND1 gene significantly reduced the binding of patient-derived autoantibodies to HeLa cells. This result indicates that the autoantigen recognized by the patient-derived autoantibody having neuropathic pain is Plexin D1.

[Experimental Example 10]
(Examination of reactivity of patient-derived autoantibodies to Plexin D1 2)
The same examination as in Experimental Example 9 was performed using serum derived from 11 patients who contained autoantibodies reactive to tissue sections of mouse dorsal root ganglia.

FIG. 20 (a) is a representative fluorescence micrograph showing the result of detecting a patient-derived autoantibody (case 5) bound to a HeLa cell into which a control siRNA was introduced. In FIG. 20A, the nucleus is stained with DAPI. The scale bar indicates 50 μm. FIG. 20 (b) is a representative fluorescence micrograph showing the result of detecting a patient-derived autoantibody (case 5) bound to a HeLa cell into which siRNA against the PLXND1 gene has been introduced. In FIG. 20B as well, nuclei are stained with DAPI. The scale bar indicates 50 μm.

FIG. 20 (c) is a graph in which the results of FIGS. 20 (a) and (b) are digitized. As a result, it was revealed that the binding of autoantibodies derived from the patient in case 5 was significantly reduced in HeLa cells into which siRNA for the PLXND1 gene was introduced.

The same examination was performed for patient-derived autoantibodies other than Case 5. As a result, similar results were obtained for 9 patient-derived autoantibodies out of 11 patient-derived autoantibodies. This result further supports that the self-antigen recognized by the patient-derived autoantibody having neuropathic pain is Plexin D1.

[Experimental Example 11]
(Study on reactivity of patient-derived autoantibodies to Plexin D1 3)
Immunoresorption experiments were conducted to examine the reactivity of patient-derived autoantibodies to Plexin D1. Specifically, a recombinant human Plexin D1 protein in which the extracellular domain portion of the PLXND1 gene is expressed in a patient-derived autoantibody (type “4160-PD”, R & D Systems, amino acid sequence is shown in SEQ ID NO: 8). Were mixed and pre-incubated. Subsequently, the fluorescent indirect antibody method using tissue sections of mouse dorsal root ganglia and mouse spinal cord was performed.

FIG. 21 (a) is a fluorescence micrograph showing the result of reacting a patient-derived autoantibody (Case 5) with a tissue section of a mouse dorsal root ganglion. The scale bar indicates 50 μm.

FIG. 21 (b) is a fluorescence micrograph showing the results of reacting a patient-derived autoantibody (Case 5) preincubated with 0.5 μg / mL Plexin D1 protein to a tissue section of a mouse dorsal root ganglion. The scale bar indicates 50 μm.

FIG. 21 (c) is a fluorescence micrograph showing the result of reacting the patient-derived autoantibody (Case 5) preincubated with 2 μg / mL Plexin D1 protein to the tissue section of the mouse dorsal root ganglion. The scale bar indicates 50 μm.

FIG. 21 (d) is a fluorescence micrograph showing the result of reacting a patient-derived autoantibody (Case 5) with a tissue section of a mouse spinal cord. The scale bar indicates 50 μm. Binding of patient-derived autoantibodies was observed at the arrowed portion.

FIG. 21 (e) is a fluorescence micrograph showing the result of reaction of a patient-derived autoantibody (Case 5) preincubated with 0.5 μg / mL Plexin D1 protein to a tissue section of a mouse spinal cord. The scale bar indicates 50 μm. An arrow is shown at the same position as in FIG.

FIG. 21 (f) is a fluorescence micrograph showing the result of reacting a patient-derived autoantibody (case 5) preincubated with 2 μg / mL Plexin D1 protein to a tissue section of a mouse spinal cord. The scale bar indicates 50 μm. An arrow is shown at the same position as in FIG.

As a result, it was confirmed that preincubation with Plexin D1 protein decreased the staining of dorsal root ganglion neurons of patient-derived autoantibodies (Case 5) in a dose-dependent manner of Plexin D1 protein. Similar results were observed with all 11 patient-derived autoantibodies.

This result further supports that autoantibodies derived from patients with neuropathic pain have specificity for Plexin D1 protein and have pathogenicity.

[Experimental example 12]
(Study on reactivity of patient-derived autoantibodies to Plexin D1 4)
Immunoresorption experiments were conducted to examine the reactivity of patient-derived autoantibodies to Plexin D1. Specifically, a recombinant human Plexin D1 protein in which the extracellular domain portion of the PLXND1 gene is expressed in a patient-derived autoantibody (type “4160-PD”, R & D Systems, amino acid sequence is shown in SEQ ID NO: 8). Were mixed and pre-incubated. Subsequently, the mouse dorsal root ganglion-derived protein extract prepared in the same manner as in Experimental Example 7 was subjected to Western blotting and detected with patient-derived autoantibodies.

FIG. 22 (a) is a photograph showing the result of detecting a self-antigen by reacting with a patient-derived autoantibody (case 1). FIG. 22 (b) is a photograph showing a result of detecting autoantigen by reacting a patient-derived autoantibody (case 1) preincubated with 0.5 μg / mL Plexin D1 protein.

FIG. 22 (c) is a photograph showing a result of detecting a self-antigen by reacting with a patient-derived autoantibody (case 2). FIG. 22 (d) is a photograph showing the result of detecting autoantigen by reacting a patient-derived autoantibody (Case 2) preincubated with 0.5 μg / mL Plexin D1 protein.

As a result, it was confirmed that the band around 220 kDa disappeared by preincubation with Plexin D1 protein. Similar results were observed with all 11 patient-derived autoantibodies.

This result further supports that autoantibodies derived from patients with neuropathic pain have specificity for Plexin D1 protein and have pathogenicity.

[Experimental Example 13]
(Study of reactivity of patient-derived autoantibodies to Plexin D1 5)
Recombinant human Plexin D1 protein was subjected to Western blotting and detected with patient-derived autoantibodies, thereby examining the reactivity of patient-derived autoantibodies to Plexin D1.

As a recombinant human Plexin D1 protein, a protein in which the extracellular domain portion of the PLXND1 gene was expressed (model “4160-PD”, R & D Systems, amino acid sequence is shown in SEQ ID NO: 8) was used. The band size of this protein was predicted to be 165 kDa to 175 kDa.

FIG. 23 shows autoantibodies derived from neuropathic pain patients whose results by the fluorescent indirect antibody method were positive ( cases 1, 2, 5), and sera derived from neuropathic pain patients whose results by the fluorescent indirect antibody method were negative It is a photograph which shows the result of having detected recombinant human Plexin D1 protein using the serum derived from a healthy person, and a commercially available anti-human Plexin D1 antibody.

As a result, a single band having a size of between 150 kDa and 250 kDa was detected by an autoantibody derived from a neuropathic pain patient whose result by the fluorescent indirect antibody method was positive and a commercially available anti-human Plexin D1 antibody. It became clear.

On the other hand, no band was detected in the serum derived from a patient with neuropathic pain and the serum derived from a healthy person who were negative by the fluorescent indirect antibody method.

The above results further support that Plexin D1 is a protein to which an autoantibody derived from a neuropathic pain patient whose result by the fluorescent indirect antibody method is positive.

The present invention can provide a technique for detecting neuropathic pain.

Claims (16)

1. Use of autoantibodies against small unmyelinated dorsal root ganglion neurons as a neuropathic pain marker.
2. The use according to claim 1, wherein the autoantibody is an anti-Plexin D1 antibody.
3. A method for detecting neuropathic pain comprising detecting autoantibodies against small unmyelinated dorsal root ganglion neurons in a blood sample from a patient, wherein the patient detects that the autoantibodies are detected. A method of showing that it is suffering from neuropathic pain.
4. Detecting autoantibodies against small unmyelinated dorsal root ganglion neurons
   Contacting a blood sample derived from a patient with dorsal root ganglion tissue or spinal dorsal horn tissue derived from a human or non-human animal, and detecting human IgG antibody bound to the dorsal root ganglion tissue or spinal dorsal horn tissue ,
   Detecting myelinated dorsal root ganglion neurons or nerve fibers thereof in the dorsal root ganglion tissue or the dorsal horn tissue of the spinal cord;
   And wherein the human IgG antibody binds to a neuron other than the myelinated dorsal root ganglion neuron, indicating that an autoantibody against a small unmyelinated dorsal root ganglion neuron has been detected. Method.
5. Detecting myelinated dorsal root ganglion neurons
   Immunostaining the dorsal root ganglion tissue with an anti-S100β antibody,
   5. The method of claim 4, wherein the neurons immunostained with the anti-S100β antibody are myelinated dorsal root ganglion neurons.
6. Detecting autoantibodies against small unmyelinated dorsal root ganglion neurons
   Contacting a blood sample derived from a patient with dorsal root ganglion tissue or spinal dorsal horn tissue derived from a human or non-human animal, and detecting human IgG antibody bound to the dorsal root ganglion tissue or spinal dorsal horn tissue ,
   Detecting unmyelinated dorsal root ganglion neurons or nerve fibers thereof in the dorsal root ganglion tissue or the dorsal horn tissue of the spinal cord;
   4. The method of claim 3, wherein binding of the human IgG antibody to the unmyelinated dorsal root ganglion neuron indicates that autoantibodies to small unmyelinated dorsal root ganglion neurons have been detected.
7. Detecting unmyelinated dorsal root ganglion neurons
   Contacting said dorsal root ganglion tissue with isolectin B4;
   The method according to claim 6, wherein the neuron to which isolectin B4 is bound is an unmyelinated dorsal root ganglion neuron.
8. The method according to any one of claims 3 to 7, wherein the human IgG antibody is an IgG2 antibody.
9. The method according to any one of claims 3 to 8, wherein the autoantibody is an anti-Plexin D1 antibody.
10. A method for detecting neuropathic pain comprising contacting a blood sample derived from a patient with a human cell or non-human animal cell expressing Plexin D1, and detecting a human IgG antibody bound to the cell,
    When the amount of the human IgG antibody detected is higher than the amount of human IgG antibody that binds to the blood sample when the blood sample is brought into contact with cells that do not express Plexin D1, A method of showing that it is suffering from disabling pain.
11. An anti-human IgG antibody;
    A detection agent for myelinated dorsal root ganglion neurons or a detection agent for unmyelinated dorsal root ganglion neurons;
    A diagnostic kit for neuropathic pain, comprising:
12. The diagnostic kit according to claim 11, wherein the anti-human IgG antibody is an anti-human IgG2 antibody.
13. The diagnostic kit according to claim 11 or 12, wherein the drug for detecting myelinated dorsal root ganglion neurons is an anti-S100β antibody.
14. The diagnostic kit according to any one of claims 11 to 13, wherein the agent for detecting an unmyelinated dorsal root ganglion neuron is isolectin B4.
15. A diagnostic kit for neuropathic pain comprising an anti-human IgG antibody and Plexin D1 protein.
16. The diagnostic kit according to claim 15, wherein the anti-human IgG antibody is an anti-human IgG2 antibody.

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