JPH02504429A - Diagnosis and treatment of rheumatoid arthritis - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] i Umachi World Mackerel and 91 1. LJL The present invention relates to the diagnosis of rheumatoid arthritis in an individual. This method uses virus The presence of a peptide or protein is associated with the development of rheumatoid arthritis in an individual It is based on the discovery that

The invention also relates to the treatment of rheumatoid arthritis by administering antiviral agents.

2. Sitting on the bench 2.1. Yutachi Edo-sama Juri Gen 2.1.1. ＋Umachi 2′ Rheumatoid arthritis (RA) is a chronic systemic disease of unknown etiology. Tissue pathology is caused by proliferation of sulochondrial cells, destruction of articular cartilage and bone, and infiltration of mononuclear cells. It is characterized as. Its serious, debilitating sequelae are caused by a type of connective tissue in the joint. It comes from destruction and destruction.

Autoimmune MRL/17 mouse strain (?Iurphy, E, D.

and Roths, J. B., 197B, “Genetic Control of Autoimmune Diseases 4. BJ, Rose, N, R,'f5, Elsevier/North-Ho lland.

New York, pp. 207-219) is a naturally occurring rheumatoid arthritis. Suffer from a inflammatory disease (Hang + LM, 1982. J.

Exp, Med, 155:1690-1701). Intra-articular in these mice The pathological changes that occur in can be divided into three stages (0'5ullivan, F, χ, - et al. 11985, Arthur, Rheum, 28: 529- 536), the first stage occurs between 7 and 13 weeks of age and involves the formation of slipped cells within the joint recess. Consists of proliferation. The second stage is a smooth thinning with an appearance similar to that of cancerous cells. Characterized by the subsequent proliferation of cysts. The first disruptive change is in the second stage. Occurs, including peripheral erosion, shortly followed by articular cartilage and meniscal cartilage A progressive destruction of occurs. The final stage is a decrease in sulcal cell proliferation and extensive cartilage destruction. , formation of scar tissue and fibrocartilage, and mononuclear and polymorphonuclear inflammatory cells in the sulcus interstitium. characterized by moderate infiltration by

Throughout the course of MRL/1 disease, there is an obvious gap between inflammatory cell infiltration and exudation and tissue destruction. There is significant dissociation. Erosion of cartilage and subchondral bone in joints looks cancerous. It was initially associated with the proliferation, adhesion and invasion of smooth lining cells, and is associated with human RA. (Fass-bender) 1. -G, 1983. C. olCo11a Re1. Res, 3:141-155) Originally non-inflammatory Similar to joint destruction. Typical joints in MRL arthritis and H)RA Erosion occurs primarily in areas adjacent to proliferated and sloughed cells, which indicates extracellular From proliferating cells that have the ability to resolve the components of the matrix, especially cartilage and bone collagen. CKrane, S, L and Amento suggesting that the enzyme is released. , E, P, + 1983. J, Rheumat, 10( Supp, 11) Near-12). In this regard, collagen's potential major Its role and regulation have been extensively studied (Harris.

E, D-i, 1984. ColCo11a Re1. Res, 4:493 -512).

Causes increased collagenase and prostaglandin E2 production by slipped cells A substance was identified and named mononuclear cell factor or MCF (Dayer, J, M.

-Ra-21981, FEBS Lett, 124:253-256).

Although many cellular interactions between cells derived from rheumatoid arthritis have been described, The exact mechanism of underlying joint destruction remains unknown. Why are slippery cells inflammatory cells? Will it proliferate without cell infiltration (0'5ullivan, F, X-14,1 985, Arthur, Rheum, 28:529-536.　Fassbe der, H.G., 1983. ColCo11a Re1. Res, 3:141-155) and why these cells begin to attach and subsequently develop into cartilage and Whether it disrupts the connective tissue matrix of bone remains a critical question in the field. there were.

The mechanism of cell adhesion is of particular interest, especially in cartilage and bone. In MRL/1 mice, proliferating smooth canal wall cells are present during the early inflammatory stage of the disease. It occurs only in areas that appear to be in close contact with the cartilage or bone matrix. Me (0' 5ullivan.

F, χ, et al., 1985. Arthur, Rheum, 28:529-536 ), the same holds true for the initial injury of human rheumatoid arthritis. Fassbender, H, G, 1983゜ColCo11a Re1. Res, 3:141-155) *These early stages are inflammatory blood Presence of neovascular vanus and production of soluble mediators and proteolytic enzymes This is clearly distinguishable from the findings observed at later stages characterized by (Ziff, M., 1983, J. Rheum) at, 10 (Supp, 11): 13-25; Dingle, J. T.

, 193B, J, Rheumat, 10 (Supp, 11): 38 -42).

Human (Wynne-Roberts, C. R., and Anderson, C.

1978, Arthur, Rheum, 7:279-286; Ghadi ally, F, N.

1980, Ultrastruct, Path, 1:249-264) , rat (Graabaek, P., M., 1984. Lab, Inv. est, 50:690-702), and mice (Link, G., and P. orte, A,, 1978° Ce1l Ti5sue Res, 187: 251-261; Link, G., and Porte, Ll. 1978. Ce1l Ti5sue Res, 185:251-261) joint normality Electron microscopy of smooth canal walls reveals type A cells (macrophage-like phagocytes) and Type B cells (more fibroblast-like cells) were identified. MRL/1 mouse ( 0'5ullivan, F, X, 4.1985. Arthur.

Rheuwl, 28:529-536) and human rheumatoid arthritis (Fa ssbender, H, c, l 1983. ColCo11a Re1.　 Res, 3:141-155), the slippery cells that proliferate have undergone cancerous transformation. It was suggested that

Epstein-Perle virus, hepatitis B virus, rubella virus, leowill and balboviruses (Brown, K., A., 1984. Natu 309:582) are closely related to the pathogenesis of rheumatoid arthritis. (Sokoloff, L., 1984. Intl. Rev. E. xp, Pathol, 262117-119: Sm1th, C, A , 1979. J, Rheumatol, 6(2):113-11 6; April 7.1984. The Lancet, pp, 772- 774; Bacon, P., A., and Tunn, E., J., 1986. + QuarterlyJ, Med,, New 5eries 6H234 ):897-899; Snyderman.

R-4, 1987, Arthritis and Rheumatism 30 (10):1191-1194). Chronic arthritis in goats Caprine arthritis-encephalitis virus (CAEV), a lentiviral member of the virus group (Crawford, T. B. i, 1980. 5science 207:997; Banks, K. L.i., 1987.　 Arthritis and Rheumatism 30(9):1046- 1053; Brassfield, ^, L-M, 1982. Arthrit is and Rheumatism 25(8):930-936).

2.1.2. 'Equine rheumatoid arthritis (referred to as 'R') Although the etiology of ``A'' was previously unknown, it is possible to treat the pathological characteristics of the disease. Various treatments have been used. Salicylates, including aspirin, have anti- It is used for its inflammatory and analgesic effects. Ibuprofen and related compounds are used in place of salicylates. Indomethacin is another anti-inflammatory, It is an analgesic and is not suitable for treatment with salicylates. Gold compounds are used for active arthritis used with the above-mentioned compounds for RA, but if the RA condition is advanced. is not always effective. Additionally, harmful side reactions may occur with gold compounds. caused by the use of objects. D-penicilla used instead of gold compounds Mins can also cause very harmful side reactions. Mild to moderate activity Symptoms of sexual RA can be treated with hydroxychloroquine. However, due to its use, Reversible retinal damage can occur. The use of corticosteroids is a short-term anti-inflammatory drug. effect on joint damage, but does not affect the progression of joint damage (Berkow, R.i. , 1982. The? ! erckManual, 779-786).

2.2. retro virus RNA1i tumor virus is -1,) D I ≦ constitutes the Hiroshinae tumor virus subfamily. to be accomplished. -p)　O+2　≦U or retroviruses are enveloped. (For a review, see Wayward, W. , S.

and Neel+ B, G, + 198L Curr, Top, Micr obiol.

T+++ muno1.91:217-276), in their replication cycle. Utilizes DNA intermediates. The virus particle is an outer membrane element derived from the host cell plasma membrane. Consists of a ribonucleoprotein core surrounded by an envelope. virus envelope The cellulose proteins of the membrane protrude from the outer N membrane.

The viral genome consists of a single-stranded RNA molecule.

All retrovirus families and subfamilies use RNA-dependent DNA polymerases or replicates through DNA intermediates as a result of the action of enzymes such as reverse transcriptase. Reproduction The cycle begins with adsorption of the virus to the cell surface, followed by invasion of the cell membrane and virus release. The shedding of the virus particles and the release of viral RNA occur. Viral RNA is next It also serves as a template for DNA synthesis by reverse transcriptase. Part of the linear DNA double strand enters the nucleus It is carried here and transformed into a covalently closed circular DNA molecule (proviral DNA). and then integrated into the host cell's chromosome DNA. built in pro Viral DNA is replicated in the host genome and is synthesized in order to be incorporated into virus particles. Produces NA, which is further processed and translated to produce viral proteins It serves as a template for synthesizing viral messenger RNA. virus particles Assembly takes place in the cytoplasm at the cell membrane. Immature particles exit the cell through the membrane. It sprouts and matures here.

The released particles can then begin a new round of dyeing.

Avian RNAAl1I tumor viruses are divided into two groups based on their pathogenicity. Ru. Viruses that cause cancer quickly can produce neoplasms within 2-4 weeks in vivo. and will cause the cells to become morphologically cancerous in vitro (Graf, T., and Beug, H., 1978, Biochem, Biophys , Acta Rev, Cancer 516:269-299), 1! arrogantly Cancer-causing retroviruses induce neoplasms after 4-12 months, and tissue culture cells do not cause cancer (purchase).

H, G, et al., 1977, 1nfect, Tnusun, 15:423-4 28). The root of the difference in pathogenicity lies in genes22, and cancer-causing retroviruses are , has oncogenic genes that are not present in the genomes of slow oncogenic viruses.

Avian leukemia virus, or ALV, is a retrovirus that slowly causes cancer. It is a type. The genome is arranged in a straight line from 5' to 3', and LLL, and It consists of engineering genes. The m gene is a structural protein or group of the virus core. encodes the specific antigen, 1 encodes reverse transcriptase, and env encodes the envelope It encodes a glutinous protein.

The equivalent of a known retroviral oncogene, namely V-Onc, is found in normal cells. , that is, research has revealed that it also exists in C-0nC. gun Oncogenic proteins encoded by genes cover various cellular locations and functions. (For a review, see Eunter, T., 1984. Sci, Am. erican 251nia 0-79), these proteins have sequence They are grouped based on similarity of features and functionality. For example, the ras protein is G It has TP-bondability and has 1 and 1 (Hempnauer, M, - H, and 5ippel, A, E, 1986, Mo1. Ce11. Biol, 6:62-69) are both nuclear proteins with DNA-binding activity. code. Some oncogenic proteins are associated with growth factors and their receptors I know what you're doing.

In NIH3T3 (mouse fibroblast) cells, ras-induced The major proteins responsible for this are cysteine proteases and cathepsins (Jos eph.

L. et al., Nucl, Ac1cls Res, 15(7):3186). H- ras-Extent of ras expression and metastasis potential in cancerous mouse fibroblasts A positive correlation was found between , and the total amount of secreted cathebusin L ( Denhardt, D-T.i., 1987. Oncogene 2:55-5 9) Mice with cancer caused by Kirsten virus (carrying the ras oncogene) The major protein excreted from the fibroblast lineage is the catalytically active alkate. It seems to be a precursor type (!'Iason, R, W, -ra- , 1987+ Bioche+++.

J, 248:449-454).

Human T cell leukemia virus (HTLV) is homologous to human T cells A group of human retroviruses. HTLV-1 and HTLV-I It is associated with several types of leukemia and lymphoma. HTLV-■ is adult hypocellular leukemia disease (Gallo, R, C, and Wong-5taal, F, 198 2. Blood 60:545; Ga1lo, R, C, 1984 in Cancer 5urveys, Vol, 3+ Franks+ L,! '1. , et al, , eds, , 0xfcrd tlniv, Press, Oxford, pp. 113-159). ing. HTLV-! I was the first fool to contract the T-cell variant of hairy cell leukemia. Identified (Kalyanaraman, V, S, etc., is, 1 982+5science 218:571). For both viruses, -+' It has the ability to cause T cells infected with yYjodan to become cancerous, and also causes other cell changes. cause (Arya, S, K, etc. 1.

1984, 5science 225:927-930, and described therein. (See references).

The HTLV-1-like retrovirus is responsible for the progressive pulmonary disease Tropical Spasticity Deficiency. isolated from a T-cell line derived from a paralyzed patient (Jacobson, S., et al. + 198L Nature 331:540-543). Data is , linking multiple sclerosis to the presence of a novel retrovirus related to HTLV-1 (Koprohski, H0+eta1., 198 5. Nature 318:154-160), but subsequent research -1 homogeneous virus involvement could not be proven (Hauser, S., L., et al-+ 1986. Nature 322:176-177; Ka rpas, A., et al., 1986. Nature 322:1 77-178).

The causative agent of AIDS is currently known as human immunodeficiency virus type I (HIV-1). previously isolated viruses of possibly the same retroviral subgroup. (Levy, J, A,, et al-+ 1984.5science 225:840) HT L V m (Gallo, R,C.

+ et al, + 1984 + 5science 224:500; P opovic, M,, dan a1. ．． 1984. 5science 224:4 97), LAV (Barre-Sinoussi+ p, l et al, + 1983. 5science 220:868; Feori no, P, l’1. , et al, 1984.5science 2 25:69), and ARV (Levy, J. A., et al. A retrovirus called 1984.5science 225:840) be.

F (3'orf gene) of HIV (HTLV-III) is an oncogene product. It has been shown that they encode proteins with sequence homology. that tamper In cooperation with the ras protein, the protein promotes GTPase, autophosphorylation and CTP- binding activity (Guy, B., et al., 1987. Natur e 330:266-269).

Pe1ton, B, K, et al, 1988 + Annals Rheu.

Diseases 47:206-209, reverse transcriptase production and HIV RA or systemic lupus erythematosus using hybridization with cDNA probes. - No evidence of virus infection could be found in patients suffering from death (SLE). However, lentiviruses that cause infection and disease are associated with RA and SLH. Lentiviruses exhibit many immunopathological features similar to those of It has been studied in relation to these diseases. Results indicate that endogenous lentiviruses and other Interviro genomic material was not demonstrated (1979. 12:234-239), lentivirus infection is transmitted horizontally. (Yanin, A, +et al-+ 1985+V irology 145:340-345), Bis, a lentivirus. Naviruses have been shown to contain a major internal structural polypeptide, P2O. child The polypeptide has group-specific antigenic determinants common to other lentiviruses, but Cancer virus and foam virus (1979 Intervirology 12:2 It has not been found in other retroviruses such as 34-239). various cormorant Nucleotide sequence analysis of the virus genome (Chiu, LM, et al. , 1985. Nature 317:366-368) is HTLV-I [ 1 is most closely related to lentivirus CAEv, while HTLV T# and H The TLV-II genome proves to be most closely related to the oncogene subfamily. Ta.

Identification of specific viruses or virus-like components as causative factors in RA There was no. In the end, the diagnostic method for RA focuses on examination of secondary pathological features. There is. Furthermore, treatment of RA may alleviate these secondary pathological features. is directed towards.

Obviously, what is needed is to treat the cause of the disease as a predisposition and to treat the RA condition. This is a better way to test whether it is present in an individual. Furthermore, R.A. A clearer understanding of the causes will result in improved treatment protocols. can be done.

3. Master's plan aborted The present invention relates to a method for diagnosing rheumatoid arthritis in an individual. This method The presence of rheumatoid peptides and proteins contributes to the development of rheumatoid arthritis in individuals. It is based on the finding that they are related. Generally speaking, the present invention provides a method for treating patients suffering from RA. It consists of an assay to diagnose RA in patients suspected of having RA.

More specifically, the invention relates to the treatment of rheumatoid arthritis with antiviral agents. Ru. It is now clear that such treatment is related to the cause of the disease. is aimed at biological particles.

4. Two plate sitting interaction The present invention will be better understood by reference to the following detailed description of the invention and the following drawings. will be more easily understood.

Figure 1 shows that HTLV-I p19 was stained with a monoclonal antibody and then F ) Slipping of an RA patient stained with TC-labeled goat anti-mouse IgG (233/3 A fresh frozen section of 1) is shown. Less than 5% of the slipped cells are located in specific intracellular and and/or membrane staining. (X 400) Figure 2 shows formalin-fixed RA A paraffin section of synovial tissue (R1892) is shown. This is a silver-sensitized immunogold staining method. stained with a motuclonal antibody against HTLV-1p19 labeled Ta. Specific staining of sloughed cells adjacent to areas of bone erosion is shown. (X250 ) Figure 3 shows typical retroviruses observed in samples from patients suffering from RA. It is an electron micrograph showing the structure. This RA patient's serum contains HTLV-1 and It was negative for antibodies against HTLV-II. (X285,000) 5. Shusatoki and Shiseishori The present invention provides a method for diagnosing rheumatoid arthritis in an individual. Main departure Ming also describes a method for treating rheumatoid arthritis by administering antiviral drugs. give.

Generally speaking, individuals with rheumatoid arthritis have HTLV-associated protein sequences. It was discovered that it has the protein sequence of a retrovirus. This tongue The protein sequences are responsible for the proliferation of sloughed cells that appear to become cancerous and the immune response of the joints. It is associated with inflammation and inflammation. These protein sequences are the protein determinants of HTLV. Consists of an epitope recognized by an antibody directed against a constant. Rheumatism Detection of HTLV-related peptides or proteins associated with arthritis is: 1) individual diagnosis of rheumatoid arthritis, and 2) treatment of rheumatoid arthritis by administration of antiviral drugs. Provides a means to treat and soothe arthritis.

More specifically, preferred embodiments of the invention relate to methods of diagnosing rheumatoid arthritis. This is because retroviruses such as HTLV proteins are produced in individuals. Peptides or proteins consisting of epitopes, such as protein epitopes By detecting.

Such a method consists of: a) obtaining a sample from an individual; b) pass the sample to an antibody directed against a retroviral protein, such that the antibody is epitopic; contacting reaction conditions capable of binding to the group; C) removing unbound antibody molecules from the sample; d) determining the presence of bound antibody; testing the sample by Here, binding of the antibody indicates the presence of rheumatoid arthritis in the individual.

In a preferred embodiment, a model directed to an epitope of HTLV A noclonal antibody was used.

Monoclonal antibodies can be labeled. Alternatively, the method can be used to It may also consist of adding additional identified binding partners.

The monoclonal antibodies used in the present invention are anti-HTLV I p19, p24 , it is preferable to choose from a group of Delicious. However, when implementing this method, Any monoclonal antibody can be used. A preferred antibody is Dupont ( [Bil] Erica, MA), anti-HTLV 1p 19, anti-HTLV I p24, anti-HTLV I [l p15/17, anti-HTLV I[I p24 and anti-HTLV I[I gp120, and anti-HTLV reverse transcriptase.

Peptides and proteins consisting of epitopes such as HTLV protein epitopes substances that 1) do not form complexes and 2) are complex with biological substances from the group mentioned above. 3) able to bind to viruses, and 4) HTLVI , HTLVI[[, novel HTLVIn-related protein or novel HTLV- It may be a protein such as I-related retrovirus. More specifically, anti- The body is) ITLV I p19, HTLV I p24, HT L V m p 15/17, HTLV I [Ip24 and HTLV Il gp120, and anti-HTLV reverse transcriptase. may be directed against HTLV proteins.

Regarding the peptide or protein detected according to this embodiment of the invention Therefore, uncomplexed proteins are ionically bonded to any other biological material, even through covalent bonds. It is a protein that is not bound by binding.

In other cases, proteins connect to other biological substances through covalent or ionic bonds. It's complex. Additionally, the protein may be a viral replication protein or a host protein. associated with proteins produced from viral genomic material inserted into the genome. Ru.

The samples assayed include sloughed cells, tissue and synovial fluid, as well as those in the vicinity of sloughed tissue and synovial fluid. This includes the meniscus and other joint components such as articular cartilage and bone near cartilage. Not limited.

In the present invention, a sample suitable for the assay is a slipped sample, where the slipped sample is selected from the group consisting of synovial cells and synovial fluid. However, affected individuals Biological samples of other origins can be used (i.e., mononuclear peripheral blood cells) ).

Assaying labeled complexes is preferably accomplished by: 1) a. ) immunofluorescence (Figure 1), b) immuno-peroxidase method, and C) silver enhancement Staining methods such as Kokin staining (Figure 2), and 2) Microstructural analysis using an electron microscope. (as shown in Figure 3). Additionally, immunocytology, immunoblotting and and ELISA (enzyme-linked immunosorbent assay) methods are also used.

In another embodiment of the invention, as a probe a receptor such as an HTLV protein is used as a probe. Using labeled nucleic acid sequences encoding lovirus proteins, five-speed j811Ω high Brid formation testing can diagnose rheumatoid arthritis in patients . Such nucleic acid probes are recognized by the Envelope Glycoprotein Award, Core Protein Award, at least one of limerase, reverse transcriptase or other retrovirus-specific proteins including, but not limited to, DNA, cDNA, or RNA sequences that may partially encode Not done.

More specifically, it is clear how to diagnose rheumatoid arthritis in patients, which consists of: (a) Isolating tissue samples from patients (b) Probes The tissue sample is incubated under conditions that allow it to hybridize to the @acid in the sample. Contacting with a nucleic acid probe specific for torovirus genes (C) Removing unbound probe: and (d) Probe high detecting hybridization, where hybridization of probes is This figure shows rheumatoid arthritis in patients. In particular, retroviral genes include HTLV genes. It's a legacy.

Alternatively, the diagnostic method may involve isolation of a nucleic acid sample from the follicle and subsequent retroviral hybridization to a nucleic acid probe specific for the gene. this Such methods are particularly suitable for Northern or Southern hypersode formation procedures or cell docking procedures. may include a tutoplot hybridization method.

Another embodiment B of the present invention is to treat patients by administering an antiviral agent to the patient. This is a method for treating rheumatoid arthritis in patients. Acyclovir is an antiviral drug. , phosphosformic acid, ribavirin, “antisense” oligodeoxynucleotides, Protease inhibitor, 2”, 3’-dideoxynucleoside, methyl Select from a portion consisting of phosphite oligonucleotides and intertyuron is desirable. Furthermore, the above 2'.

3'-dideoxy nucleoside is 3'-azido-2', 3'-dideoxy selected from the group consisting of thymisin and 2',3''-dideoxycytidine. Ru.

6. Emperor” L± 6.1. A monochrome that is used against retroviruses. ・1 6.1.1., Fireflies of 6.1.1.1. exemption J Lt rainbow method Cryos Prepared by tat sectioning. Air-dried sections were treated with appropriate monoclonal antibodies. Incubated with For controls, the sections were prepared with immunoglobulin from pre-immune serum. Incubated with globulin (1 g). in a warming chamber at room temperature. After incubation for 30 minutes, sections were placed in phosphate buffered saline (PBS, pH 7.4). In a second step, the sections were rinsed three times with Layer with 0 diluted fluorescein-isothiocyanate (FITC) for 30 minutes. . Finally, the slides are thoroughly washed to remove non-specifically attached reagents. and sealed under a coverslip with 90% glycerol/10% PBS. Ru. Localization of the staining was carried out using a Lei system equipped with a two-filter system for FITC. Observe and photograph using a tz-fluorescence microscope.

6.1.1.2. Peroxygenase-injected paraffin sections are treated with xylene and Remove paraffin in alcohol. Tryptic digestion (0,1 [/d, Tris -HCL, 60 min), the sections were treated with 0.3% hydrogen peroxide to remove any endogenous peroxide. Oxidase activity is inactivated and rinsed with 0.05% saponin (2 x 15 minutes each).

Sections were then pretreated with normal serum diluted 1:5 in 0.2% BSA/PBS. , washed and partially incubated with the first antibody at 4°C. After washing, VE CTASTAIN-ABC Kit (Vector Iab, Inc.) Using Bur-1ingame, CA), write () tsU et al, , 198LJ, Histochem, Cytochem 29:557- 580), the ABC technique is used.

6.1.1.3. To the Ph feeling (TGGS) Baraffin sections are as described above. Remove paraffin and hydrate. Sections were treated with 5% heat inactivation inhibiting serum (goat) for 30 min. incubate for 1 minute, followed by partial incubation at 4°C with monoclonal antibody. do. Thoroughly washed slides were soaked in PBS, pH 7.3, containing 0.1% BSA. : Total complex antibody diluted to 40 (Auroprobe L?l Goat Antibody Mouse I gG(Janssen, Life Sci, Products, 01en +Be1g1u+++)) and incubate for 2 hours at room temperature.

Freshly prepared silver enhancement reagent (Inten) in PBS (and after washing with distilled water) SE 11 (Janssen, Life Sci, Products, Cover the sections with 01en+Belgium) and monitor the reaction under a light microscope. - to do.

After 5-15 minutes, the reaction was stopped by washing with distilled water, and the sections were treated with hematoxylineosin. After staining and cleaning, fix it on a slide.

6-1.2. □ When immunoelectron microscopy is performed on a sulcus biopsy, the specimen is two razor blades, Or cut to a thickness of 0.2-0.5 mm using a cryostat. They are, 1% glucose in 0.16M cacodylate buffer, pH 7.4 for 20 min at 4°C. Fixed with taraldehyde [R, Fleisch-majer et al. J, Invest, Dermat, 75:]89J91 (1980) , 0.15M) Wash several times with Lis-HCl buffer, pH 7.5 to remove excess After removing the rutaraldehyde, it is stored at 4°C in the same sofa. monoku Incubation with local antibodies was performed at 1:5 in PBS, pH 7.2. 24 hours at 4°C in the presence of ferritin-labeled rabbit anti-mouse Ig antibody diluted to Time is done. The sample was 0.1M) Lis-HCl buffer, pH 7.5. Rinse for 24 hours at 4°C using 0.16M cacodylate pamphor, p) ( Washed three times with 7.5 and 3% glycol in 0.16M cacodylate 82fur 1 pH 7.4. Fixed with rutaraldehyde for 2 hours. The control sample was rabbit anti-mouse Ig ferri Treat only with Chin-labeled antibody. The samples were post-fixed with osmium tetroxide and graded. It is embedded in araldite through alcohol. That block is LKB Ultradome - cut into one piece with 4 microtome, about 500-600 people (O ngström) thick specimens may be left unstained or treated with uranyl acetate. Stained with saturated solution. The grid is Hitachi HLf-12A or P h1llips can be examined using a 300 electron microscope.

6.1.3. Menkofu plate! Other cells, e.g. isolated sulcal membrane cells or mononuclear peripheral blood cells, were incubated with phosphate buffered cells. Fix with 4% paraformaldehyde for 2 hours at 4°C with one change of solution [Ga y et al,, ColCo11a Re1. Res, 1:370-3 77 (1981)].

Next, 4% cells! Test at 4°C for 36 hours with PBS containing dirt, and do not change it many times. Wash thoroughly.

The final wash was performed for 1 hour with PBS containing 4% soap and 5% glycerol. cormorant. The cell pellet was then placed in an oven with a cork or plastic support backing. CT Freeze Methylbutane (isopentane) placed in the ground in a small container of liquid nitrogen. Freeze them quickly by submerging them in a wide-mouthed jar. frozen The cells are then wrapped in aluminum foil and stored at 120 degrees in an airtight container. . Frozen sections 8 μm thick were cut and mounted on albumin-coated slides. , air dry for at least 5 minutes. Next, the slides were soaked in ice-cold NaBHa PBS. solution (10 mg/100 m) and leave for 1 hour with - changes. After this step, Wash the slides in PBS for three 30 minute changes at 4°C.

Cell sections were incubated with suitable monochrome cells in a humidified container overnight at 4°C or for 2 hours at room temperature. react with null antibody. Wash slides thoroughly with PBS, then incubate for an additional 2 hours. and a second antibody (goat or rabbit anti-mouse Ig).

This is followed by washing with cold PBS and washing with Fab-peroxidase-anti-peroxidase. A third antibody treatment is performed using Fab-PAP for 3 hours. Fab-PA The PlS solution was removed by washing with PBS, and the tissue sections were washed with 40 μm of 3,3-diaminoben. Zidine tetrahydrochloride and 15μ! 150d of 0°1M) containing 5% H2O2, Incubate for 15-18 minutes at pH 7.6. Next, put the slide in cold PB. Wash with S and stain with 1% osmium tetroxide for 1 hour at room temperature. stained slides were rinsed again with cold PBS, dehydrated with acetone, and embedded in Malaglass (70%). This creates ultra-thin sections for examination using an electron microscope.

6.2. Im Plow-in Protein immunoplot analysis is based on antibody reactivity observed in immunofluorescence studies. This is done to confirm that the Primary cultured cleft membrane cells were supplemented with 10% heat-inactivated fetal calf serum. The cells are grown in R.P.M.T-1640 medium containing supplements. Adhesive strips from duplicate culture dishes The cells are processed as follows: (1) one culture dish is washed with PBS; Cells are detached by treatment with 0.25% trypsin in medium. Collected cells are cultured Counted to determine total number of cells per dish. (2) Duplicate cell culture dishes The other side was washed with PBS without trypsinization, and the cells were directly injected with 5DS-polymer. In acrylamide gel electrophoresis sample buffer (0.1% SDS/4M urea) Collect at a final concentration of 10' cells per sample buffer. Possible? 8 hypercellular proteins (equivalent to 10S cells) were electrophoresed on a polyacrylamide gel. Dynamically separated and transferred to nitrocellulose. To suppress nonspecific antibody binding, Therefore, each plot contains 1% bovine serum albumin (BSA). Shaking in liquid Incubate. Monoclonal antibodies were diluted with 1% BSA solution and plotted. to allow antibody binding. The bound antibody is peroxidase - Incubation with conjugated goat-anti-mouse antibody (also diluted in 1% BSA solution) and visualized by 4-chloro-1-naphthol. double series One plot shows the amount of total protein transferred, stained with amide black solution. and visualize molecular weight markers.

Highly purified retrovirus protein stock solution was added to 0.02M NazCO 3 (pH 910) and plated in a 96-well microtiter plate (Dy Coat the dilution on Natech (1 mmulon) overnight. Next well was added with 0.05% Tween 20.8 and 1% Methyl to remove unbound proteins. Wash three times with phosphate buffered saline (PBS-Vienna) containing luorate. P Rates were blocked with 0.5% bovine serum albumin in PBS (PBS-BSA) followed by washing with PBS-Tween.

6.3.2.21 March 11 Serum from patients with rheumatoid arthritis is obtained by blood collection. as a negative control , using a single lot of pooled human serum.

Patient serum was diluted with PBS 1:1. 1: 10. 1: Diluted to 100 and 50μ of each dilution! was added to duplicate wells and incubated for 1 hour at 37°C. Bate. Next, the wells were washed three times with PBS-Tween, 1:50 Horseradish peroxidase (HRP) conjugated goat anti-human diluted to 0 Immunoglobulin (New England Nuclear Screenin) g System NEI-602) Add 50uR to each well and Incubate for 1 hour at 7°C.

After washing 5 times to remove unbound peroxidase-conjugated antibody, 50μ! freshly prepared substrate (0-phenylenediamine) was added to the wells and incubated at 37°C for 15 min. Incubate. Next, the reaction was stopped with 4.5M sulfuric acid, and 492 Automated ELISA reader (Titer Tek Mult) iscan).

Alternatively, s1-labeled sheep anti-human immunoglobulin (specific activity, 5-20. ! IC/μg, Amersham, ArlinHeight s, north,) and fractionated to give 50.000 counts per well. , and then incubate the resulting wells at 37°C for another hour. Do not combine The radioactivity was removed by extensive washing with PBS plus 0.05% Tween. The radioactivity in each well was counted using a Backman 5500 gamma counter. Count.

b, 4. + Retrovirus of rheumatoid arthritis, which is the arrow of the horse world Diagnosis of HTLV-related pathologies in particular has proven successful.

In one study, retroviral products were shown to be effective in patients with early-stage destructive rheumatoid arthritis. HTLV I p19 and This was confirmed by immunofluorescence using a monoclonal antibody against P24. another In the study, expression of retroviral products was and p24, and monoclonal against HT V-I p15/17 Antibody-based immunofluorescence was performed on fresh, unfixed, slipped tissue samples of ca. Observed at 0%. Retroviral production is recommended in patients without rheumatoid arthritis disease. No manifestation of any substance was observed. Silver-enhanced immunogold staining was fixed in formalin and paraffinized. 40% of the patient's tissue was All rheumatoid arthritis stained flank muscles tested No specific staining was detected in the tissue.

Antibodies against HTLV I or HIV-1 are Depending on the stun immunoblotting technique, RA! ! , not observed in human serum. won.

However, in some cases of RA, serum ELISA or for immunoelectron microscopy despite negative results by stamplot Examination of the sulcus membrane tissue by immunogold technique showed the presence of typical retroviral structures. (Figure 3). The structure with a diameter of about 65 nm is HI V-T p15/ It contains an electron-dense core surrounded by a double membrane labeled with an antibody specific for 17. Ta.

Monoclonal antibody specific for HIV-1 reverse transcriptase (Cellular P products, Inc. + Buffalo, NY). reverse transcriptase was detected in approximately 70% of the smooth tissue from RA patients.

6.5.One night stay! In the present invention, 3'-azido-2',3-dideoxysilane is used for the treatment of rheumatoid arthritis. Antiviral agents such as cythymidine (Zidovudine) are used. this The drug has many human or animal receptors, such as HTLV-1 and HTLV-I. It has been shown to be active in vitro against viruses and reverse transcription enzymes. Retroviruses such as those related to primary activity or p24 core antigen (gag protein) It has been specifically investigated for its cytopathological effects on antigens. 0.13. ! /g/mL of zidovudine (HTLV-mB) and When used together, no expression of p24 core antigen could be detected.

Zidovucline is administered orally. Currently, this drug is not available in the United States. Although there is no commercially available dosage form for parenteral administration, the drug can also be administered by intravenous infusion. is given. The initial oral dose of Ziclovudine is 200 Dosing every 4 hours six times a day is currently recommended; however, this recommendation is limited. The total volume and administration timetable have not been fully established. do not have. This dose corresponds to 2.9 kg/kg every 4 hours for a 70 kg patient. do. In clinical trials, zidovudine is usually administered at doses of 250 -5,9 ■/kg), but up to 15 ■/kg every 4 hours. Oral administration was used for a short period of time (i.e., 4 weeks) in a limited number of patients.

Ribavirin has also been tried in the treatment of RA. This drug stimulates T cells (T-white blood cells) has been shown to stimulate and also inhibit antigen-induced proliferation in a dose-dependent manner . Additionally, ribavirin has been shown to be effective against many RNA viruses, including members of the Retroviridae family, and H. Shows antiviral activity against TLV-111 subfamily.

Ribavirin was administered using Viratek Particulate 9 Atomizer (SPAC) Model 5PAG-2. It is administered only by nasal or oral inhalation as a solution. 6g Nori Babi Phosphorus was reconstituted as a sterile powder by dissolving 50 to 100 mL of sterile water. do. This solution is then transferred to a sterile 500-Ill flask. this flask becomes the 5PAG-2 sprayer glue reservoir. This solution is further diluted with sterile water for inhalation. 20TI with diluted final volume of 300! g/mH solution. Contains additives Diluents cannot be used to reconstitute babilin. In addition, rehabilitation ? The solution must be free of any particulate matter or discoloration prior to its administration.

Once the solution is properly reconstituted, nebulization therapy begins with oxygen from the 5PAC-2 nebulizer. Better done with a hood, face mask, or oxygen tent .

20 μ/d of vapilin solution was used as the starting solution in the 5PAC-2 reservoir. When the 5PAC-2 sprayer is in use, the 5PAC-2 sprayer produces a mist containing approximately 190 μg of glue babilin per liter. Release.

The recommended administration of ribavirin doses reaches the respiratory tract according to the following formula: Ribavirin concentration) xO, 7 (percentage of inhaled amount settled in the respiratory tract) More specifically, inhalation treatment with the drug at a rate of 12.5 L of mist per minute It was applied for 16-18 hours. Then, on the third and third days of treatment, 12 hours a day (-daily allowance) Administration was continued for 4 hours (three times for 4 hours) on the fourth day (last day) of treatment.

Acyclovir is another candidate for RA treatment.

This drug is used to treat inflammation and pain in immunocompromised individuals during oral acyclovir treatment. It appeared to reduce the risk of cancer, prevent new lesions, and promote healing of lesions. Furthermore, reeds The main therapeutic effect of Clovia is to halt the progression of osteoarthritis. Seem. Although the use of oral acyclovir has only recently been used, This may 1) prevent new lesions, 2) possibly reduce the duration of pain, and 3) ) It is thought to bring about healing of lesions.

Administration of acyclovir sodium requires slow intravenous infusion. It will be done. Administration by rapid intravenous infusion (less than 10 minutes) or rapid intravenous injection should be performed. do not have.

To reconstitute the acyclovir sodium, add 500 μl of it to 10 aft. Add to distilled water for injection. Alternatively, add 500 μ of acyclovir sodium to benzyl Add to paraben-free bacteriostatic water for injection containing alcohol. These reconstructions The solution should be used within 12 hours.

Before administering oral acyclovir, shake the solution to ensure complete dissolution of the drug. must be done. Typically, the amount of reconstitution solution required is 50-125 d. diluted with intravenous infusion solution. Slow intravenous infusion with dilute solution , to reduce the risk of adverse renal effects that may occur if the infusion is done for less than one hour. To avoid this, do it for over an hour.

Oral administration of 200 μ acyclovir every 4 hours while awake, 5 times a day for 10 days. This is the recommended adult dosage. At the time of recurrence, the recommended adult dose is 20 0■ three times a day for up to 26 weeks. In some cases, 200 ■ - 5 times a day 26 Doses of up to a week are required. Intermittent treatment of recurrence is 200 ■ awake This would require 5 doses for 5 days - 4 hours every day.

Normal renal function (i.e. creatinine clearance 1.73rd per 50m) The intravenous dose of acyclovir for adults with 5 cm/kg every 8 hours (- day 15■/kg) from 7th to 10th day.

Furthermore, the present invention inhibits the adhesion of cancerous lateral abdominal canal wall cells to cartilage and bone. patient treatment, which consists of administering to the patient a drug that suppresses the erosion and destruction of the joint due to The present invention relates to a method for treating rheumatoid arthritis in patients. The drug acts on cell adhesion receptors. This is the binding peptide sequence.

Each of the above embodiments and any antiviral agents or treatment modalities described herein are solely inventions. As such, the invention is not intended to be limited in scope by these. I don't intend that. Furthermore, all the factors that determine whether an individual has RA symptoms methods for treating RA with functionally equivalent antiviral agents as described earlier in the text. Any method of treatment is intended to be within the scope of this invention. Shown and described here Various modifications of the invention, in addition to those described above, will become apparent to those skilled in the art from the foregoing description and accompanying specification. It will become clear to the person. Such modifications are within the scope of the appended claims. intend to.

FIG, I FIG. 2 FIG.3 international search report ■ PCT/IJSB91022U9 xt:ach*er+7 to, Form PCT/IPEA/2105eco n65heetヱC1assifxcatxon 43S15-7. 2Eh 4361506.525,530. 801.　 1tosi 514/2. 44-:il:r+-,,C1,+4+ 16 1K 31/70. 37102: Cl2Q l/68. 70: GO IN337548.553.564 Medlir+e+ Rheuratoid (w) arthriti s and fretrcvirn or mvor LAV or RI V) ! ’, eclir+e and Biosis: arthriti? and 5ynovi (2wlcell? and fattae h? or a6hern or adhesi? l　anareee pt mouth;? PCT/US89102219

Claims (1)

[Claims] 1. A method for diagnosing rheumatoid arthritis in an individual having: a) Obtaining a sample from an individual; b) pass the sample to an antibody directed against a retroviral protein, such that the antibody is epitopic; contact under reaction conditions that allow for bonding to the group; c) removing unbound antibody molecules from the sample; and d) testing the sample for the presence of bound antibodies; Here, binding of the antibody indicates the presence of rheumatoid arthritis in the individual. 2. 2. The method of claim 1, wherein the antibody is labeled. 3. without further addition of a labeled partner that specifically binds to the antibody. The method according to claim 1. 4. The method of claim 1, wherein said antibody is a monoclonal antibody. 5. The antibody is anti-HTLV Ip19, anti-HTLV Ip24, anti-HTL V III p15/17, anti-HTLV VIII p24, anti-HTLV II I gp120 and anti-HTLV reverse transcriptase of the claim selected from the group consisting of Method of scope 1. 6. Claim 1, wherein said retroviral protein is an HTLV protein. the method of. 7. The HTLV protein is HTLV I p19, HTLV1 I p24, HTLV III p15/17, HTLVVIII p24, HTLV III Claims selected from the group consisting of gp120 and anti-HTLV reverse transcriptase The method of Section 6. 8. A synovial membrane sample, wherein the sample is selected from the group consisting of synovial cells, synovial tissue, and synovial fluid. The method according to claim 1. 9. 2. The method of claim 1, wherein said sample is peripheral blood cells. 10. (d) where the step is carried out by ultrastructural analysis using an electron microscope; Method of scope 1. 11. The ultrastructural analysis by electron microscopy was performed by immunoelectron microscopy. 10. The method of claim 10. 12. 2. The method of claim 1, wherein step (d) is carried out by immunofluorescence analysis. 13. Claim 1, wherein step (d) is carried out by an immunoperoxidase method. Section method. 14. Claim 1, wherein the step (d) is carried out by silver-sensitized immunogold staining. Law. 15. The method of claim 1, wherein step (d) is performed by immunocytology. 16. Claim 1, wherein the step (d) is carried out by immunoplotting. the method of. 17. 2. The method of claim 1, wherein step (d) is carried out by ELISA. 18. Method for diagnosing rheumatoid arthritis in patients with: (a) Obtaining a tissue sample from a patient (b) hybridizing the probe to the nucleic acid in the tissue sample of step (a); The tissue sample from step (a) is treated with a retroviral gene-specific Contacting with a nucleic acid probe (c) removing the unbound step (b) probe; and (d) detecting hybridization of a probe, wherein the probe The hybridization of is indicative of rheumatoid arthritis in the patient. 19. Claims in which the retroviral gene in step (b) is an HTLV gene. The method of Section 18. 20. Method for diagnosing rheumatoid arthritis in patients with: (a) Obtaining a tissue sample from a patient (b) isolating a nucleic acid sample from the tissue sample in step (a); (c) hybridize the probe to the isolated nucleic acid sample of interest in step (b); The nucleic acid sample of step (b) is subjected to the formation of retroviral mRNA sequences. (d) contacting the unbound (c) step-specific nucleic acid probe; removing the pipe; and (e) detecting said hybridization of said probe of step (c); wherein said hybridization of said probe is associated with rheumatoid joints in a patient. Showing flame. 21. Method for diagnosing rheumatoid arthritis in patients with: (a) Obtaining a tissue sample from a patient (b) isolating a nucleic acid sample from the tissue sample in step (a); (c) hybridize the probe to the isolated nucleic acid sample of interest in step (b); The nucleic acid sample of step (b) is subjected to the formation of retroviral mRNA sequences. Contacting with a column-specific nucleic acid probe 22. The retrovirus at stage (c) 21. The method of claim 20, wherein the HTLV gene is an HTLV gene. 23. The retroviral mRNA sequence in step (c) is an HTLV mRNA sequence. 22. The method of claim 21. 24. To reduce rheumatoid arthritis in a patient, administer an effective amount of an anti-inflammatory drug to the patient. treatment of a patient with rheumatoid arthritis, comprising administering an antiviral agent. composition. 25. The antiviral agents include acyclovir, phosphonoformic acid, ribavirin, and oligodeoxynucleotide, protease inhibitor, 2',3'- A group consisting of dideoxynucleosides and methylphosphite oligonucleotides 24. The configuration of claim 23 selected from. 26. The 2',3'-dideoxynucleoside is 3'-azido 2',3'-dideoxynucleoside. selected from the group consisting of deoxythymidine and 2',3'-dideoxycytidine; The structure of claim 25. 27. The 3'-azido-2',3'-dideoxythymidine is from about 2 mg of the 3'-azido-2',3'-dideoxythymidine to about 15 administered in doses up to mg 3'-azido-2',3'-dideoxythymidine , the configuration of claim 26. 28. It inhibits the adhesion of cancerous synovial canal wall cells to cartilage and bone, thereby damaging the joints. The patient's treatment consists of administering an effective amount of a drug capable of inhibiting erosion and destruction of the Composition of treatment of rheumatoid arthritis in patients. 29. Claim 1, wherein the agent is a peptide sequence that binds to a cell adhesion receptor. Structure of Section 28.