Early diagnosis of Mycoplasma pneumoniae-infection has been eager to be established, and specific and sensitive diagnostic method using Mycoplasma pneumoniae lipid-antigen made it possible.
We already have established ELISA using chemically synthesized lipid-antigen of Mycoplasma pneumoniae. Using this ELISA, we are able to know the infection of Mycoplasma pneumoniae. Within three days after onset of fever, we are able to detect Mycoplasma pneumoniae infection in children seeing IgM which specifically increase in these patients. Also we are able to see the incerese of IgG in patients with Mycoplasma pneumoniae infection who are previously infected and the IgM does not increase and IgG are increasing.
PA: more than 320 or 640 is positive. GGL Glc-type ELISA: more than 2 or 3 is positive, and quantitative.
Glycoglycerophospholipid, antibody thereagainst, and method for detecting mycoplasma Document Type and Number:United States Patent 6376203http://www.freepatentsonline.com/6376203.html
(WO/2007/145362) GLYCEROGLYCOLIPID ANTIGEN OF MYCOPLASMA PNEUMONIAE
(WO/2007/145361) LIPID ANTIGEN DETECTION METHOD USING MASS SPECTROMETER
Chemically synthesized lipid-antigens are available.
Anti-Mycoplasma fermentans lipid-antigen monoclonal antibody is available.
Human B dendritic cell line is available.
It is well known that M. pneumoniae causes 40% or more cases of community-acquired pneumonia and as many as 18% of cases requiring hospitalization in children (1). Recent epidemiologic studies indicated that M. pneumoniae are frequently involved as causative agents in upper or lower respiratory tract infections including common colds, pharyngitis, bronchitis, wheezing, and extrapulmonary disease in
children aged less than 5 yr as well as in school ages and throughout the year with periodic epidemics at 3-7 yr intervals (2). M. pneumoniae had been isolated from the respiratory tract of 20-50% of asthmatic patients with acute exacerbations and patients with chronic stable asthma (2). (1) Waites KB, Talkington DF. Mycoplasma pneumoniae and its role as a humanpathogen. Clin Microbiol Rev 2004; 17: 697-728. (2) Principi N, Esposito S. Emerging role of Mycoplasma pneumoniae and Chlamydia pneumoniae in paediatric respiratory-tract infections. Lancet Infect Dis 2001; 1: 334-44.
Mycoplasma have been reported as pathogens of autoimmune diseases, rheumatic diseases, asthma. However, it was difficult to fix the pathogenesis , because there were not reliable diagnostic tools to identify or detect them. M Bio Technology Inc. have succeeded to determine the structures of the specific-lipid antigens of them and also succeeded to synthesize them completely by chemical synthesis. Base on these high-technology, we are developing diagnostic tools and aiming novel type of drugs.
Mycoplasma lipid antigens are useful as bio-markers of the diseases. These lipid antigens are useful to measure the antibodies against them. Further more, it is lead to the development of novel type of drugs according to the analysis of the molecular mechanism and using it as a screening system.
Mycoplasmas have been reported as pathogens of autoimmune diseases, rheumatic diseases, athma, so on. However, it was difficult to fix the pathogenesis, because there were not reliable diagnostic tools to identify or detect them. We have succeeded to determine the structures of the specific lipid-antigens and succeeded to synthesize them. Based on these technology, we developed diagnostic tools.
Mycoplasma fermentans glycolipid-antigen as a pathogen as a pathogen of rheumatoid arthritis.
Biochemical and Biophysical Research Communications 369 (2008) 561–566
Corresponding author : Dr. Kazuhiro Matsuda
Mycoplasma fermentans has been suspected as one of the causative pathogenic microorganisms of rheumatoid arthritis (RA) however, the pathogenic mechanism is still unclear. We, previously, reported that glycolipid-antigens (GGPL-I and III) are the major antigens of M. fermentans. Monoclonal antibody against the GGPL-III could detect the existence of the GGPL-III antigens in synovial tissues from
RA patients. GGPL-III antigens were detected in 38.1% (32/84) of RA patient’s tissues, but not in osteoarthritis (OA) and normal synovial tissues. Immunoelectron microscopy revealed that a part of GGPL-III antigens are located at endoplasmic reticulum. GGPL-III significantly induced TNF-a and IL-6 production from peripheral blood mononulear cells, and also proliferation of synovial fibroblasts. Further study is necessary to prove that M. fermentans is a causative microorganism of RA; however, the new mechanisms of disease pathogenesis provides hope for the development of effective and safe immunotherapeutic strategies based on the lipid-antigen, GGPL-III, in the near future.
Rheumatoid arthritis is a chronic multisystem disease of unknown cause. Microvascular injury and an increase in the number of synovial lining cells appear to be the earliest lesions in rheumatoid synovitis. Subsequently, synovial lining cells are increased along with perivascular infiltration with mononuclear cells. Light-microscopic examination discloses a characteristic constellation of features,
which include hyperplasia and hypertrophy of the synovial lining cells; focal or segmental vascular changes, including microvascular injury, thrombosis, and neovascularization;
edema; and infiltration with mononuclear cells, often collected into aggregates around small blood
vessels. The predominant infiltrating cell is the T lymphocyte. Besides the accumulation of T cells, rheumatoidsynovitis is also characterized by the infiltration of variable numbers of B cells and antibody-producing plasma cells. Finally, the synovial fibroblasts in RA may be activated, and may degrade components of the articular matrix. These activated fibroblasts are particularly prominent
in the lining layer, and at the interface with bone and cartilage.
The rheumatoid synovium is characterized by the presence of a number of secreted products of activated lymphocytes, macrophages, and fibroblasts. The local production of these cytokines and chemokines appears to account for many of the pathologic and clinical manifestations of RA.
The cause of RA remains unknown. It has been suggested that RA might be a manifestation of the response to an infectious agent in a genetically susceptible host. A number of possible causative agents have been suggested, including Mycoplasma, Epstein-Barr virus (EBV), cytomegalovirus,
parvovirus, and rubella virus, but convincing evidence that these or other infectious agents cause RA has not emerged. One possibility is that there is a persistent infection of articular structures or retention of microbial products in the synovial tissues that generated a chronic inflammatory response.
Mycoplasma fermentans has been reported as a pathogenic microorganism of chronic inflammatory diseases, such as RA, chronic fatigue syndrome, fibromyalgia, and neurological diseases [1–8]. However, this association has been difficult to prove. Previously, we reported that phosphocholine-containing glycoglycerolipids (GGPLs: GGPL-I and GGPL-III) are the lipid-antigens of M. fermentans . The lipid-antigens are expressed in M. fermentans specifically, and these lipid-antigens are the major lipid-antigens of M. fermentans . Matsuda et al. found
and determined the complete structures of GGPL-I and GGPL-III as 6-O-phosphocholine-a-glucopyranosyl-(1-3)-1, 2-diacyl-sn-glycerol, and 100-phosphocholine 200-amino
dihydroxypropane-300-phospho-60-a-glucopyranosyl-(1-3)-1,2-diacyl-glycerol, respectively [10–13]. Based on the structures and bioactivities of GGPLs, we have proposed the hypothetical role of M. fermentans in the pathogenesis [14–16]. Because GGPLs have strong immunogenicity, they
may play roles as immunodisturbing agents in cell functions such as inflammation and cell differentiation . We established monoclonal antibody which specifically recognizes
the GGPL-III structure .
In this study, we demonstrated a direct evidence which suggested that M. fermentans is regarded as the pathogen of RA.
 M.H. Williams, J. Brostoff, I.M. Roitt, Possible role of Mycoplasma fermentans in pathogenesis of rheumatoid arthritis, Lancet 2 (1970) 277–280.
 C.B. Gilroy, A. Keat, D. Taylor-Robinson, The prevalence of Mycoplasma fermentans in patients with inflammatory arthritides, Rheumatology (Oxford) 40 (2001) 1355–1358.  S. Horowitz, B. Evinson, A. Borer, J. Horowitz, Mycoplasma fermentans in rheumatoid arthritis and other inflammatory arthritides, J. Rheumatol. 27 (2000) 2747–2753.
 J. Haier, M. Nasralla, A.R. Franco, G.L. Nicolson, Detection of mycoplasmal infections in blood of patients with rheumatoid arthritis, Rheumatology (Oxford) 38 (1999) 504–509.
 T. Sohaeverbeke, C.B. Gilroy, C. Bebear, J. Dehais, D. Taylor-Robinson, Mycoplasma fermentans in joints of patients with rheumatoid arthritis and other joint disorders, Lancet 347 (1996) 1418.
 T. Schaeverbeke, C.B. Gilroy, C. Bebear, J. Dehais, D. Taylor-Robinson, Mycoplasma fermentans, but not M. penetrans, detected by PCR assays in synovium from patients with rheumatoid arthritis and
other rheumatic disorders, J. Clin. Pathol. 49 (1996) 824–828.
 J. Nijs, G.L. Nicolson, P. De Becker, D. Coomans, K. De Meirleir, High prevalence of Mycoplasma infections among European chronic fatigue syndrome patients. Examination of four Mycoplasma species
in blood of chronic fatigue syndrome patients, FEMS Immunol. Med. Microbiol. 34 (2002) 209–214.
 G.L. Nicolson, M.Y. Nasralla, J. Haier, J. Pomfret, High frequency of systemic mycoplasmal infections in Gulf War veterans and civilians with Amyotrophic Lateral Sclerosis (ALS), J. Clin. Neurosci. 9 (2002)
 K. Matsuda, J.L. Li, R. Harasawa, N. Yamamoto, Phosphocholinecontaining glycoglycerolipids (GGPL-I and GGPL-III) are speciesspecificmajor immunodeterminants of Mycoplasma fermentans,
Biochem. Biophys. Res. Commun. 233 (1997) 644–649.
 K. Matsuda, I. Ishizuka, T. Kasama, S. Handa, N. Yamamoto, T.Taki, Structure of a novel phosphocholine-containing aminoglycoglycerolipid of Mycoplasma fermentans, Biochim. Biophys. Acta 1349 (1997) 1–12.
 K. Matsuda, T. Kasama, I. Ishizuka, S. Handa, N. Yamamoto, T.Taki, Structure of a novel phosphocholine-containing glycoglycerolipid from Mycoplasma fermentans, J. Biol. Chem. 269 (1994) 33123–33128.
 Y. Nishida, H. Ohrui, H. Meguro, M. Ishizawa, K. Matsuda, T. Taki, S. Handa, N. Yamamoto, Synthesis and absolute configuration of 6-O-phosphocholine-a-D-glucopyranosyl glycerolipid isolated from
HTLV-I-infected cell lines, Tetrahedron Lett. 35 (1994) 5465–5468.
 Y. Nishida, Y. Takamori, H. Ohrui, I. Ishizuka, K. Matsuda, K. Kobayashi, Synthesis and absolute configuration of a novel aminoglycoglycerolipid, species-specific major immunodeterminant of
Mycoplasma fermentans, Tetrahedron Lett. 40 (1999) 2371–2374.
 K. Matsuda, Phosphocholine-containing glycoglycerolipids of Mycoplasma fermentans as a pathogen of rheumatoid arthritis: Possible role of Mycoplasma fermentans GGPLs in the pathogenesis of neuroendocrine-immune abnormalities, Recent Res. Devel. Neurosci. 1 (2004)
 K. Matsuda, M. Saito, N. Yamamoto, Antigens:Lipids, Encyclopedia of Life Science, first ed., Nature Publishing Group, 2001.
 K. Matsuda, second ed.Antigens:Lipids, Encyclopedia of Life Science, vol. 1, Wiley, 2007.
 S. Schutze, K. Potthoff, T.Machleidt, D. Berkovic, K. Wiegmann, M. Kronke, TNF activates NF-kappa B by phosphatidylcholine-specific phospholipase C-induced ‘‘acidic” sphingomyelin breakdown, Cell 71(5) (1992) 765–776.
 K. Matsuda, J.L. Li, S. Ichinose, R. Harasawa, M. Saito, N.Yamamoto, Monoclonal antibody against Mycoplasma fermentansspecific aminoglycoglycerolipid, Microbiol. Immunol. 44 (2000) 695–702.
Phospholipids and glycolipids are the major components of biomembranes. Immune responses against them are implicated in several human diseases and in recognition by higher organisms of microbial infections. Structural analysis of lipid antigens is important for understanding the mechanisms of molecular interactions and the pathogenesis of immune abnormalities.
Kazuhiro Matsuda, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan
Copyright © 2006 John Wiley & Sons, Ltd. Article Online Posting Date: January 27, 2006
Mycoplasma lipid-antigens as immunoregulators in autoimmune diseases
Kazuhiro Matsuda 1,2
1. National Institute of Advanced Industrial Science and Technology (AIST) AIST Tokyo Waterfront, Tokyo, Japan, 135-0064
2. Research and Development Department, M Bio Technology Incorporation, Tokyo, Japan, 135-8073
For the study of the innate immune response in infectious diseases and autoimmunity, as well as tumors, natural killer T (NKT) cells have become a major focus. Recognizing phospholipid and glycolipid antigens presented by CD1 molecules the novel T lymphocytes produce both Th1 and Th2 cytokines. Most mouse and human B cells express CD1d. Interactions between CD1d expressed on B cells and CD1d-restricted T cells may play a role in determining amount, isotype, and specificity of the antibodies produced.
Mycoplasmas have been reported as pathogens of autoimmune diseases, rheumatic diseases, asthma, neurological disorders, so on. Anti-glycolipid, such as Gal-cer, antibodies are reported to be present in the patients of autoimmune diseases with preceding mycoplasma infection. Mycoplasma lipid-antigens induce anti-mycoplasma antibodies, and they also act as autoantibodies. We have succeeded to determine the structures of the lipid-antigens, and revealed molecular mimicry. These lipid-antigens may be useful for the analysis of the signaling mechanism bridging innate and acquired immunity.
1. Kawahito, Y., Ichinose, S., Sano, H., Tsubouchi, Y., Kohno, M., Yoswhikawa, T., Tokunaga, D., Hojo, T., Harawsawa, R., Nakano, T., Matsuda K. Mycoplasma fermentans glycolipid-antigen as a pathogen of rheumatoid arthritis. Biochemical and Biophysical Communication 369 : 561-566 (2008)
2. Matsuda, K. Lipid antigen. Encyclopedia of Life Science 2nd edition, Nature publishing group (in press)
3. Matsuda, K. Phosphocholine-containing glycoglycerolipids of Mycoplasma fermentans as a pathogen of rheumatoid arthritis: Possible role of Mycoplasma fermentans GGPLs in the pathogenesis of neuroendocrine-immune abnormalities. Recent. Res. Devel. Neurosci. 1: 15-23 (2004)
4. Matsuda, K., Saito, M., Yamamoto, N. Lipid antigen Encyclopedia of Life Science, Nature publishing group, vol.1 pp748-755. (2002)
5. Matsuda, K., Li, J.L., Shizuko, I., Ryo, H., Saito, M., Yamamoto, N. Monoclonal antibody against Mycoplasma fermentans-specific aminoglycoglycerolipid. Microbiol. Immunol. 44: 695-702 (2000)
6. Matsuda, K., Ishizuka, I., Kasama, T., Handa, S., Yamamoto, T., Taki, T. Structure of phosphocholine-containing aminoglycoglycerolipids: a novel lipid antigen of Mycoplasma fermentans. Biochim. Biophys. Acta 1349: 1-12 (1997)
7. Matsuda, K., Li, J.L., Harasawa, R., Yamamoto, N. Phosphocholine-containing glycoglycerolipids (GGPL-I and GGPL-III) are species-specific major immunodeterminants of Mycoplasma fermentans. Biochem. Biophys. Res. Com. 233: 644-649 (1997)
8. Matsuda, K., Harasawa, R., Li, J.L., Kasama, T., Taki, T., Handa, S., Yamamoto, N. Identification of phosphocholine-containing glycoglycerolipids purified from Mycoplasma fermentans-infected human helper T-cell culture as components of M. fermentans. Microbiolo. Immunol. 39: 307-313 (1995)
9. Matsuda, K., Kasama, T., Ishizuka, I., Handa, S., Yamamoto, N., Taki, T. Structure of a novel phosphocholine-containing glycoglycerolipid from Mycoplasma fermentans. J. Biol. Chem. 269: 33123-33128 (1994)
10. Matsuda, S., Matsuda, K., Ito, Y. Separation of phospholipids and glycolipids using analytical toroidal-coil countercurrent chromatography. II. Comparison of the hydrophobicity between Mycoplasma fermentans and human-brain lipids. J. Liq. Chrom. & Rel. Technol. 26: 1135-1147 (2003)
11. Shingu, Y., Nishida, Y., Dohi, K., Matsuda, K., Kobayashi, K. Converient access to halide ion-catalyzed alpha-glycosylation free from noxious fumes at the donor synthesis. J. Carbohydr. Chem. 21: 605-611 (2002)
12. Matsuda, K., Matsuda, S., Saito, M., Ito, Y. Separation of phospholipids and glycolipids using analytical toroidal-coil countercurrent chromatography. I. Separation of human brain lipids. J. Liq. Chrom. & Rel. Technol. 25: 1267-1281 (2002)
13. Nishida, Y., Takamori, Y., Ohrui, H., Ishizuka, I., Matsuda, K., Kobayashi, K. Synthesis and absolute configulation of a novel aminoglycoglycerolipid, species-specific immunodeterminant of Mycoplasma fermentans. Tetrahedron lett. 40: 2371-2374 (1999)
14. Shingu, Y., Nishida, Y., Dohi, K., Matsuda, K., Kobayashi, K. Converient access to halide ion-catalyzed alpha-glycosylation free from noxious fumes at the donor synthesis. J. Carbohydr. Chem. 21: 605-611 (2002)
15. Nishida, Y., Takamori, Y., Ohrui, H., Ishizuka, I., Matsuda, K., Kobayashi, K. Synthesis and absolute configulation of a novel aminoglycoglycerolipid, species-specific immunodeterminant of Mycoplasma fermentans. Tetrahedron lett. 40: 2371-2374 (1999)
16. Nishida, Y., Takamori, Y., Matsuda, K., Ohrui, H., Yamada, T., Kobayashi, K. Synthesis of artificial glycoconjugate polymer carrying 6-O-phosphocholine--D-glucopyranoside, biological active segment of main cell membrane glycolipids of Mycoplasma fermentans. J. Carbohydr. Chem. 18: 65-72 (1999)
17. Li, J.L., Matsuda, K., Takagi, M., Yamamoto, N. Detection of serum antibodies against phosphocholine-containing aminoglycoglycerolipid specific to Mycoplasma fermentans in HIV-1 infected individual. J. Immunol. Methods 208: 103-113 (1997)
18. Nishida, Y., Ohrui, H., Meguro, H., Ishizawa, M., Matsuda, K., Taki, T., Handa, S., Yamamoto, N. Synthesis and absolute configuration of 6-O-phosphocholine--D-glucopyranosyl glycerolipid isolated from HTLV-I-infected cell lines. Tetrahedron Lett. 35: 5465-5468 (1994)
19. Matsuda, K., Taki, T., Hamanaka, S., Kasama, T., Rokukawa, C., Handa, S., Yamamoto, N. Glycosphingolipid compositions of human T-lymphotropic virus type I (HTLV-I) and human immunodeficiency virus (HIV)-infected cell lines. Biochim Biophys Acta 1168: 123-129 (1993)
20. Matsuda, K., Yamamoto, N., Kaneko, T., Iwahashi, M., Hashimoto, S., Araki, K. Hypercalcemia and serum TNF-α in T-cell leukaemia. Lancet 335(8696): 1032 (1990)
21. Kitamura, K., Matsuda, K., Ide, M., Tokunaga, T., Honda, M. A fluorescence sandwich ELISA for detecting soluble and cell-associated human interleukin-2. J. Immunol. Methods 121: 281-288 (1990)
22. Honda, M., Kitamura, K., Matsuda, K., Yokota, Y., Yamamoto, N., Mitsuyasu, R., Chermann, J.C., Tokunaga, T. Soluble IL-2 receptor in AIDS: correlation of its serum level with the classification of HIV-induced diseases and its characterization. J. Immunol. 142: 4248-4255 (1989)