Monoclonal Antibody against Mycoplasma fermentans‐Specific ...

8 downloads 2454 Views 9MB Size Report
cobiology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan ... genesis of rheumatoid arthritis. ..... We call attention to its.
Microbiol.

Monoclonal Antibody fermentans-Specific

Immunol., 44(8), 695-702, 2000

against Mycoplasma Aminoglycoglycerolipid

Kazuhiro Matsuda*,4 4, Jin-Liang Li1, Shizuko Masaki Saito4, and Naoki Yamamoto'

Ichinose2,

Ryo Harasawa3,

1Department

of Microbiology, and 'Laboratory for Electron Microscopy; Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8519, Japan, 3Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-8654, Japan, and 4Virology and Glycobiology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan Received

November

18, 1999; in revised

form, May

16,2000.

Acceped

May

17,2000

Abstract: Previously, we reported that Mycoplasma fermentans has specific antigens (phosphocholinecontaining glycoglycerolipids: GGPL-I and GGPL-III) and discussed the possibility of their pathogenic role. In this paper, we report the characterization of a monoclonal antibody (MF-III-1) specific to GGPL-III (phosphocholine-containing aminoglycoglycerolipid) using methods of electron microscopy, immunofluorescence cell surface staining, laser scanning microscopy, immunoelectron microscopy, and thin-layer chromatography immunostaining. The MF-III-1 antibody specifically recognized M. fermentans attached to the surface of HTLV-I-infectedhuman helper T-cells,and it did not cross-react with other lipids nor with human T-cell antigens. Since MF-III-1 distinguishes GGPL-III from GGPL-I, the binding site may include a serinol (2-amino-1,3-propanediol)residue of GGPL-III. MF-III-1 is useful for the in vitro study of M. fermentans, and may also be useful as a tool for the study of the involvement of M. fermentans in human diseases. Key words: Monoclonal antibody, HTLV-I-infected noelectron microscopy

human helper T-cell, Mycoplasma fermentans,

Mycoplasma fermentans is suspected as a pathogen of rheumatoid arthritis (RA) and other human diseases (1, 4, 16, 27-29, 34, 35). M. fermentans is frequently isolated from the synovial fluid of rheumatoid arthritis patients (27, 29, 34, 35) and exhibits agglutinating activity toward erythrocytes. These observations suggest the possibility that M. fermentans has a role in the pathogenesis of rheumatoid arthritis. Recently, interest in M. fermentans has increased because it is frequently isolated from, or detected in, various tissues and blood of AIDS patients (1, 4, 12, 16) and has been implicated in the disruption of the immune system. M. fermentans also has high affinity for HTLV-I-infected T-cells (26). Autoimmune-like diseases are often seen in HTLV-Iinfected patients (11), therefore, it is suspected that M. fermentans may have some pathogenic' role in these patients. This, however, is still obscure because of the difficulty in detecting M. fermentans clinically. Previously, we reported that M. fermentans contained phosphocholine-containing glycoglycerolipids (GGPLs: GGPL-I and GGPL-III) (17), and determined the com-

plete

structures

of

GGPL-I

and

Immu-

GGPL-III

as

phocholine-ƒ¿-glucopyranosyl-(1'-3)-1, erol

and

respectively

structures

of

GGPL-I main

and

GGPL-III

lipid

that

(CRP)

of

the

increases

19,

M.

in

of

to a phosphocholine-containing

to

form

system the

a

and

chemical in Fig.

and

(2,

20).

inflam-

has

the

and

the

ability moi-

complement

cell

death.

Thus,

M. fermentans

have

the

and

have

a role

complexes

It has pro-

infection,

activates

might

are

C-reactive

CRP

1.

and

glycolipid

that

of

The

human to

9),

inflammation

antigens

immune

(8,

complex

induces

GGPL

form

damage

-

shown expressed

response

to ety

are

fermentans

amount

or

2-diacyl-sn

22-25).

specifically

mation bind

tissue

'-3)-1,

GGPL-III

are

antigens

reported

tein

(18,

GGPL-I

and

been

2 "-aminodihydroxypropane-

'-2-glucopyranosyl-(1

glycerol,

the

2-diacyl-sn-glyc-

1 "-phosphocholine,

3 "-phospho-6

6-O-phos-

ability in

to the

pathogenesis. In ization

this

paper, of

the

we mouse

report MoAb

establishment (MF-III-1)

and that

characterspecifically

Abbreviations: FBS, fetal bovine serum; GGPL, phosphocholine-containing glycoglycerolipid; MoAb, monoclonal antibody; PBS, phosphate-buffered saline; TLC, thin-layer chromatography. The abbreviations for gangliosides (GM, GM], GD1a and GD1b) are according to Svennerholm's nomenclature

*Address correspondence to Dr . Kazuhiro Matsuda, Virology and Glycobiology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. Fax: 03-3543-2181. E-mail: [email protected]

(31). 695

696

K. MATSUDA

Fig. 1. Structure of GGPL-III, GGPL-I, phosphatidylcholine,

ET AL

platelet-activating

recognizes GGPL-III. The results of electron microscopy, cell-surface staining and immune electron microscopic and thin-layer chromatography immunostaining analyses are presented. Also, GGPL-III availability in the in vivo or in vitro studies of M. fermentans is discussed.

factor (PAF) and sphingomyelin.

MT-4 cells, and the purified GGPL-III have been described previously (17, 19, 22, 23). Lipids of various mycoplasma strains were extracted by the method of Bligh and Dyer (3).

Immunization of mice and hybridoma preparation. BALB/c

mice

purified

Materials

and Methods

at

37

C

culture.

in

a PPLO

Mich.,

U.S.A.)

serum

(FBS),

McLean,

5%

concentration) by

washed

red.

was

with

fetal

dextrose

mycoplasmas

10,000 •~

g

phosphate-buffered

and

for

(final

saline

har-

min,

and

(PBS),

pH

7.2. fermentans

obtained and

T.

the

same

had

been Lipid

fermentans

from

PG18 G.H.

Sasaki

Cassell

(Tokyo),

strain

as

cultured extraction GGPL

strains

(#1)

(Birmingham,

respectively. PG

strain

in

our

laboratories

strain

(#2) Ala.,

PG18

the

and

and

(#2),

but for

The

21,

the

was

PG18

(#3)

1 year.

Lipids

of

and

fermentans-infected

RPMI

M.

The FBS,

harvested and

detoxified

GGPL-III

Research

from

Immuno

at each

Strepto-

Chem

(20

pig)

injected

On

injections

according

Research

was

immunization. s.c.

Inc., from

Chem

endotoxin

(Ribi

PBS,

Tesque dimycolate

Immuno

performed

TLC

of

days

14

GGPL-III.

to the

for

staining.

Kohler

the

incubated

used

for

using

second

were

and

the

lipids

supplemented

were with

and

1 00

100

of

2

10%

Um]

three ill

of

M.

MT-4

for

with

washed

first

screening.

cultured

streptomycin cells

was

M. fermentans-infected

104/m1)

medium

( 1 4)

immunostaining

used

(30 •~

of

with

(Nacalai

GGPL-III

was

1640

emulsified

with

(10).

and

(17)

immunized

trehalose

received

using

100 ƒÊg/m1 the

mice

Cell-surface cells

of

R595

was

fermentans

of

of

method

ELISA

was

(Ribi

(s.c.)

all

Milstein

tg

emulsified

Hybridization

U.S.A.)

(#3)

purification. M.

were

mg

minnesota

screening, M.

50

subcutaneously and

were 30

50

were

adjuvant

phlei

and

coccus Inc.).

penicillin,

GGPL-III

Japan),

Inc.),

bovine

female)

incomplete

Mycobacterium

Laboratories,

of

percent

The at

grown Detroit,

(v/v)

(Flow Uml

One

added.

centrifugation

twice

10%

extract 1,000

were

Laboratories,

with

yeast

phenol

strains

(Difco

U.S.A.),

(w/v)

vested

broth

supplemented

Va.,

0.002%

Mycoplasma

weeks,

GGPL-III.

Freund's Kyoto,

Mycoplasma

(4

times

MF-III-1

of

days (v/v)

in FBS,

penicillin. in PBS-1% hybridoma

a

MONOCLONAL

ANTIBODY

culture supernatant liquid ( X 10) at 37 C for 1 hr. The cells were then washed three times in PBS-1% FBS, and incubated with 100 ill of fluorescein-5-isothiocyanate (FITC) conjugated goat anti-mouse IgG (heavy and light chains) polyclonal antibodies (Bethyl Laboratories Inc., Montgomery, Ala., U.S.A.) ( X 200) at 37 C for 1 hr. The cells were then washed three times with PBS-1% FBS. Localization of GGPL-III antigen was detected by fluorescence microscopy or by a laser scanning microscope, LSM-GB200 (Olympus, Tokyo). Electron microscopy. M. fermentans-infected MT-4 cells were layered over a glass cover slide pretreated with poly-L-lysine (50 14/m1) for 2 hr at 37 C, and washed 3 times with PBS. The cells were fixed for 2 hr in 2.5% glutaraldehyde in 0.2 M PBS, stained with 1% 050, in 0.2MPBS at 4 C for 2 hr. Cells were dehydrated by exposure to increasing concentrations of ethanol. For the preparation of samples for scanning electron microscopy, the cells were dried at a critical point and sputter-coated with a thin layer of gold palladium. Samples were examined in a Hitachi S-700 (Hitachinaka, Japan) at an accelerating voltage of 15 kV. Micrographs were recorded on a Polaroid type 55 positive/negative film. For the preparation of samples for transmitted immune electron microscopy, the cells were processed for conventional Epon embedding. Ultra-thin sections were cut and transferred to nickel grids. The sections were then treated with 0.5% H20, in water at 25 C for 5 min. The grids were incubated with PBS-1% FBS for 30 min to block the non-specific attachment of antibodies to residual glutaraldehyde. After washing three times with PBS-1% FBS, samples were reacted with anti-GGPL-III MoAb or a control solution at 4 C overnight and then washed three times with PBS-1% FBS. The grids were incubated with a gold-conjugated goat anti-mouse IgG and IgM antibody (the gold particle size was approximately 20 nm) (Biocell Research Laboratories, Cardiff, U.K.) ( X 10). The samples were washed extensively with PBS-1% FBS three times and finally washed with distilled water. The sections were stained with saturated aqueous uranyl acetate for 10 min and washed with distilled water. The sections were then 'viewed with a Hitachi H-600 electron microscope at an accelerating voltage of 75 kV. Thin-layer chromatography (TLC). The developing solvent was a mixture of chloroform:methano1:0.2% aqueous CaC1, (50:45: 10, by volume). Lipids were separated on a high-performance TLC (HPTLC)-plate (Merck, Darmstadt, Germany). For the detection of lipids, the following spray reagents were used: orcinol reagent (30) for glycolipids and Dittmer's reagent (5) for

AGAINST

GGPL-III

697

phospholipids. Immunostaining of lipids on an HPTLC-plate was performed as described previously (21). Horseradish peroxidase (HRPO)-conjugated goat anti-mouse IgG (heavy and light chains) (Bethyl Laboratories Inc.) was used for the second antibody. An antigen band was visualized with a Konica immunostaining HRP kit (Konica Co., Ltd., Tokyo). Results

and Discussion

We established a monoclonal antibody MF-III-1 against the phosphocholine-containing aminoglycoglycerolipid (GGPL-III) purified from M. fermentans. To demonstrate the specificity and usefulness of the MF-III1, we carried out the following studies. The scanning electron microscopy shows the M. fermentans attached to the surface of HTLV-I-infected human helper T-cells (MT-4 cells) (Fig. 2A). The attached microorganisms were round, rod, or four-leafclover shaped (Fig. 2B). However, it is difficult to distinguish M. fermentans from the microvilli of the MT-4 cell. Then, the MF-III-1 was used to stain the MT-4 cells (Fig. 3). The staining intensity of each M. fermentansattached cell varied (Fig. 3C), and this difference may be attributed to the amount of M. fermentans attached to the MT-4 cell surface. M. fermentans-infected MT-4 cells were not stained by the preimmune mouse IgG fraction (Fig. 3B). M. fermentans-free MT-4 cells were not stained (data not shown). The location of the GGPL-III antigen on the cell was analyzed using laser scanning microscopy, which showed that GGPL-III antigens were stained along the cell surface (Fig. 3C). Scattered or accumulated M. fermentans bodies can be seen. On the right side, the localization of M. fermentans is shown schematically. The result of confocal microscopy suggested that M. fermentans were attached to the MT-4 cell surface. To confirm this, immunoelectron microscopy was carried out. This MF-III-1 antibody could also detect M. fermentans (Fig. 3, D and E). The gold particles show the existence of GGPL-III antigens. The gold particles accumulated on the M. fermentans attached to the surface of an MT-4 cell. However, there are no particles on the MT-4 cell cytoplasm and nucleus. The result clearly shows that the MF-III-1 antibody recognizes M. fermentans specifically. Figure 3, C and D shows a different area. Figure 3C shows the locus where the M. fermentans membrane appears to attach to the MT-4 cell membrane. Figure 3D may correspond to the clover or rod shape (detail in Fig. 2B). A few gold particles could be seen on the surfaces of MT-4 cells. We investigated a chemical structure specific to the

K. MATSUDA

698

A

FT AL

B

Fig. 2. Scanning electron microscopy (SEM) of a M.fermentans-infected MT-4 cell. (A) Many mycoplasma attached to the surface of MT-4 cells. Bar= 1 pm. (B) Microorganisms with round, rod or four-leaf-clover shapes can be seen. Bar=l pm.

GGPL-III by using TLC-immunostaining methods to determine the specificity of the MF-III-1 antibody. Purified GGPL-III was developed on TLC and stained as phospholipids and glycolipids (Fig. 4A). The phospholipid (lane 1) and glycolipid (lane 3) patterns of the M. fermentans GGPL strain show that GGPL-I and GGPLIII are the main phospholipids and glycolipids. The GGPL-III band could not be detected by Dittmer's staining (Fig. 4D, lane 4). However, MF-III-1 could detect GGPL-III (Fig. 4E, lane 4). GGPL-III was not detected in M. arthritidis (Fig. 4E, lane 8). A few faint bands that were also detected by MF-III-1 may have the same antigenic determinant as GGPL-III (GGPL-III-related antigens). The lipid extract (Fig. 4B, lane 1 and 3) and the purified GGPL-III (Fig. 4B, lane 2 and 4) were used as antigens. The MF-III-1 antibody recognized the GGPLIII band and purified GGPL-III (Fig. 4B, lane 1 and 2). However, normal mouse serum did not react with any lipids of M. fermentans (Fig. 4B, lane 3 and 4). To determine whether the MF-III-1 MoAb crossreacted with human cell lipids, we analyzed the neutral lipid fraction (containing phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, etc.) and the acidic lipid fraction (containing phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, etc.) of M. fermentans-free MT-4 cells. The acidic lipid fraction also contained glycolipids including GM1a,GM2 and GM, (21). They did not react with MF-III-1 (data not shown). Then, the neutral (Fig. 4C, lane 1 and 3) and acidic (Fig. 4C, lane 2 and 4) lipids of M. fermentans-infected

MT-4 cells were analyzed. The phospholipid components were visualized by Dittmer's staining (Fig. 4C, lanes 1 and 2), and MF-III-1 specifically recognized the GGPL-III band (Fig. 4C, lane 3). Trace bands were also recognized (Fig. 4C, lane 4). We have previously determined the structure of GGPL-III (18, 23, 25). Although GGPL-III has a structural similarity to phosphatidylcholine, sphingomyelin, platelet-activating factor (PAF) and GGPL-I (Fig. 1), the MF-III-1 MoAb does not cross-react with these compounds or with the glycolipids of human helper Tcells. All of the above contain phosphorylcholine residues which have hydrophilic head groups, while the ceramide or diacylglycerol (DAG) groups form hydrophobic tail groups. Thus, the amino group of GGPL-III is important because it is recognized by MFIII-1 MoAb. Because the recognition of MF-III-1 is highly specific, the minor bands recognized by the MFIII-1 MoAb may have the same epitope as GGPL-III. Recently, M. fermentans has been shown both to have oncogenic activity (32, 37) and to activate the hypothalamo-pituitary adrenal axis (33). This microorganism contains a large amount of unique phosphocholine-containing glycophospholipids (20). We call attention to its choline residue which has the potential to interact with cholinergic neurons or with the choline metabolic pathway (e.g., sphingomyelin pathway) and act as a pathogen. M. fermentans may also be involved in the pathogenesis of AIDS dementia (36). MF-III-1 may be a useful tool for confirming our hypothesis that the presence of this

MONOCLONAL

ANTIBODY

AGAINST

699

GGPL-III

A

B

C

E

D

Fig.

3. Immunofluorescence

1 MoAb

(A)

and

ic representation in

M. fermentans-infected

a normal of the

staining mouse manner MT-4

of M. IgG

of

the cells

fermentans.

fraction

(B).

attachment (D

and

Optical Confocal of M.

E).

Bar

=

microscopy laser

fermentans 5 ƒÊm

to (A,

microorganism has a role in these human diseases. Phosphatidylcholine, sphingomyelin and platelet-activating factor (PAF) have important roles in cell signal transduction. Because of their molecular similarity, it is

of

microscopic

B

MT-4

and

M.

fermentans-infected

image

C).

of an

cell

(right)

Bar

=0.5 ƒÊm

MT-4

intensively

(C).

stained

Immune (D

and

electron

cells cell

stained (left)

microscopy

with and

the of

MF-IIIschematGGPL-III

E).

postulated that GGPLs have biological functions affecting signaling systems. Previously, we reported that GGPLs have strong antigenicity and species-specific antigens of this mycoplasma (20) and that they are active

700

K. MATSUDA

ET AL

D

A

E B

C

Fig. 4. (A) Thin-layer chromatography of lipids from M.fermentans GGPL strain (lanes 1 and 3) and purified GGPL-III (lanes 2 and 4). Development was performed with a mixture of chloroform:methano1:0.2%CaCl2(55:45:I0, by volume). Developed lipids were visualized with Dittmer's (lanes I and 2), and orcinol reagent (lanes 3 and 4). (B) TLC immunostaining of M. fermentans lipids using an antiGGPL-III monoclonal antibody (MF-III-1). Lipids from M.fermentans GGPL strain (lanes I and 3) and purified GGPL-III (lanes 2 and 4) were developed with a mixture of chloroform:methanol:0.2% CaC12(55:45:10, by volume). Lipids were detected with anti-GGPLIII MoAb (MF-III- 1) (lanes I and 2) and non-immunized rabbit serum (lanes 3 and 4). (C) TLC immunostaining of M.fermentans-infected human helper T-cell lipids using anti-GGPL-III monoclonal antibody (MF-III-1). Neutral (lanes 1 and 3) and acidic (lanes 2 and 4) lipids extracted from M.fermentans-infected MT-4 cells were developed with a mixture of chloroform:methanol:0.2% CaCl2 (55:45:10, by volume). Developed lipids were visualized with Dittmer's (lanes 1 and 2) and TLC immunostaining using the MF-III-1 antibody (lanes 3 and 4). (D) TLC of lipids of various mycoplasma strains. Lipids were extracted from: M. fennentans GGPL strain (lane I), M.fermentans incognitus strain (lane 2), M.,fermentans F7 strain (lane 3), M.fermentans PG18 strain (#1) (lane 4), M.fennentans PG18 (#2) (lane 5), M. fermentans PGI8 (#3) (lane 6), M. fermentans F1 strain (lane 7) and M. arthritidis strain (lane 8). Developed lipids were visualized by Dittmer staining. (E) TLC immunostaining of lipids of various mycoplasma strains using MF-III-1 antibody. Lipids were extracted from: M. fennentans GGPL strain (lane 1),M.fermentans incognitus strain (lane 2), M.fermentans F7 strain (lane 3), M.fermentans PG18 strain (#1) (lane 4), M.fermentans PG18 (#2) (lane 5), M.fermentans PG18 (#3) (lane 6), M.fermentans Fl strain (lane 7), and M. arthritidis strain (lane 8).

in modulating interleukin 6 production. We have evidence that lipids of M. fermentans can induce HIV from latently HIV-infected macrophages. It was found that MF-III-1 antibody can recognize and neutralize this activity (paper in preparation). M. fermentans has also been reported to be capable of fusing with T-cell and peripheral lymphocytes (7, 13, 15). Our hypothesis for pathogenesis is as follows: M. fermentans invades the lesion and contributes to the acute inflammation. The GGPLs and other antigens may form immune complexes which persist and continue to stimulate the immune response resulting in more tissue damage. RNA or DNA are digested with RNAase or DNAase; therefore, PCR or isolation methods cannot be used to detect these microorganisms. There is also a report that primers used for the detection of M. fermentans in AIDS was not specific (6). On the other hand, GGPL-III antigen is stable and specific to GGPL-III structure. As a result, the

MF-III-1 MoAb may be more useful for the histochemical or immunoelectron microscopic analysis of pathological tissues. MF-III-1 may be an important tool in helping to determine the mechanisms underlying these processes. The fact that the structure of the GGPL-III antigen is well characterized is an advantage for the further study of molecular mechanisms. We would like to thank Tsuguo Sasaki (Department of Safety Research on Biologics, National Institute of Health, Tokyo) for M. fermentans Fl and F7 strains and Shyh-Ching Lo (Division of Molecular Pathobiology, Department of Infectious and Parasitic Disease Pathology, Armed Forces Institute of Pathology, Washington, D. C.) for the M. fermentans incognitus strain. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture, and the Ministry of Health and Welfare, Japan, 3rd Field of Science and a grant from Seikagaku Co., Ltd.

MONOCLONAL

ANTIBODY

References

1) Bebear, C., De Barbeyrac, B., Clerc, M.T., Renaudin, H., Fleury, H.J., Dupon, M., Ragnaud, J.M., and Morlat, P. 1993. Mycoplasmas in HIV-1 seropositive patients. Lancet 341: 758-759. 2) Ben-Menachem, G., Wagner, F., Zahringer, U., Rietschel, E.T., and Rottem, S. 1997. Antibody response to MfGL-II, a phosphocholine-containing major lipid of Mycoplasma fermentans membranes. FEMS Microbiol. Lett. 154: 363-369. 3) Bligh, E.G., and Dyer, W.J. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37: 911-917. 4) Dawson, M.S., Hayes, M.M., Wang, R.Y., Armstrong, D., Kundsin, R.B., and Lo, S.C. 1993. Detection and isolation of Mycoplasma fermentans from urine of human immunodeficiency virus type 1-infected patients. Arch. Pathol. Lab. Med. 117: 511-514. 5) Dittmer, J.C., and Lester, R.L. 1964. A simple, specific spray for the detection of phospholipids on thin-layer chromatograms. J. Lipid Res. 5: 126-127. 6) Ditty, S.E., Connolly, M.A., Li, B.J., and Lo, S.C. 1998. Mycoplasma orale has a sequence to the insertion-like sequence of M. fermentans. Moll. Cell. Probes 13: 183185. 7) Franzoso, G., Dimitrov, D.S., Blumenthal, R., Barile, M.F., and Rottem, S. 1992. Fusion of Mycoplasma fermentans strain incognitus with T-lymphocytes. FEBS Lett. 303: 251-254. 8) Kagan, G.Y., Volfovich, Y.V.,Zilfyan, A.V., Zheverzh, I.V., and Gamova, N.A. 1982. Experimental polyarthritis induced by Mycoplasma fermentans in rabbits. Zh. Mikrobiol. Epidemiol. Immunobiol. 3: 107-110. 9) Kaplan, M.H., and Volanakis, J.E. 1974. Interaction of Creactive protein complexes with the complement system. I. Consumption of human complement associated with the reaction of C-reactive protein with pneumococcal C-polysaccharide and with choline phosphatides, lecithin and sphingomyelin. J. Immunol. 112: 283-289. 10) Kohler, G., and Milstein, C. 1976. Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Eur. J. Immunol. 6: 511-519. 11) Kuwabara, H., Katanaka, J., Nagai, M., Uda, H., Hojo, W., Yamada, A., Mild, H., Takeuchi, H., and Teranishi, K. 1993. Human T lymphotropic virus type I associated myelopathy with pulmonary and cutaneous lesions. J. Clin. Pathol. 46: 273-275. 12) Lemaitre, M., Guetard, D., Henin, Y., Montagnier, L., and Zerial, A. 1990. Protective activity of tetracycline analogs against the cytopathic effect of the human immunodeficiency viruses in CEM cells. Res. Virol. 141: 5-16. 13) Lemaitre, M., Henin, Y., Destouesse, F, Ferrieux, C., Montagnier, L., and Blanchard, A. 1992. Role of mycoplasma infection in the cytopathic effect induced by human immunodeficiency virus type 1 in infected cell lines. Infect. Immun. 60: 742-748. 14) Li, J.L., Matsuda, K., Takagi, M., and Yamamoto, N. 1997. Detection of serum antibodies against phosphocholine-con-

AGAINST

GGPL-III

701

taining aminoglycoglycerolipid specific to Mycoplasma fermentans in HIV-1 infected individuals. J. Immunol. Methods 208: 103-113. 15) Lo, S.C., Shih, J.W., Newton, P.B.d, Wong, D.M., Hayes, M.M., Benish, J.R., Wear, D.J., and Wang, R.Y. 1989. Viruslike infectious agent (VLIA) is a novel pathogenic mycoplasma: Mycoplasma incognitus. Am. J. Trop. Med. Hyg. 41: 586-600. 16) Lo, S.C., Tsai, S., Benish, J.R., Shih, J.W., Wear, D.J., and Wong, D.M. 1991. Enhancement of HIV-1 cytocidal effects in CD4-1- lymphocytes by the AIDS-associated mycoplasma. Science 251: 1074-1076. 17) Matsuda, K., Harasawa, R., Li, J.-L., Kasama, T., Taki, T., Handa, S., and Yamamoto, N. 1995. Identification of phosphocholine-containing glycoglycerolipids purified from Mycoplasma fermentans-infected human helper T-cell culture as components of M. fermentans. Microbiol. Immunol. 39: 307-313. 18) Matsuda, K., Ishizuka, I., Kasama, T., Handa, S., Yamamoto, N., and Taki, T. 1997. Structure of a novel phosphocholine-containing aminoglycoglycerolipids of Mycoplasma fermentans. Biochim. Biophys. Acta 1349: 1-12. 19) Matsuda, K., Kasama, T., Ishizuka, I., Handa, S., Yamamoto, N., and Taki, T. 1994. Structure of a novel phosphocholine-containing glycoglycerolipid from Mycoplasma fermentans. J. Biol. Chem. 269: 33123-33128. 20) Matsuda, K., Li, J.-L., Harasawa, R., Taki, T., Handa, S., and Yamamoto, N. 1997. Phosphocholine-containing glyco-

21)

22)

23)

24)

25)

26)

27)

glycerolipids (GGPL-I and GGPL-III) are species-specific major immunodeterminants of Mycoplasma fermentans. Biochem. Biophys. Res. Commun. 233: 644-649. Matsuda, K., Taki, T., Hamanaka, S., Kasama, T., Rokukawa, C., Handa, S., and Yamamoto, N. 1993. Glycosphingolipid compositions of human T-lymphotropic virus type I (HTLVI) and human immunodeficiency virus (HIV)-infected cell lines. Biochim. Biophys. Acta 1168: 123-129. Matsuda, K., Taki, T., Kasama, T., Ishizuka, I., Handa, S., and Yamamoto, N. 1993. Occurrence of a novel glycolipid containing phosphocholine in HTLV-I-infected cells. Glycoconjugate J. 10: 340. Matsuda, K., laid, T., Kasama, T., Ishizuka, I., Handa, S., and Yamamoto, N. 1995. Characterization of a phosphocholinecontaining aminoglycoglycerolipid: a major lipid antigen of Mycoplasma fermentans. Glycoconjugate J. 12: 423. Nishida, Y., Ohrui, H., Meguro, H., Ishizawa, M., Matsuda, K., Taki, T., Handa, S., and Yamamoto, N. 1994. Synthesis and absolute configuration of 6-O-phosphocholine-a-D-glucopyranosyl glycerolipid isolated from HTLV-I-infected cell lines. Tetrahedron Lett. 35: 5465-5468. Nishida, Y., Takamori, Y., Ohrui, H., Ishizuka, I., Matsuda, K., and Kobayashi, K. 1999. Synthesis and absolute configuration of a novel aminoglycoglycerolipid, species-specific major immunodeterminant of Mycoplasma fermentans. Tetrahedron Lett. 40: 2371-2374. Phillips, D.M., Pearce, P.R., Tan, X., and Zacharopoulos, V.R. 1992. Association of mycoplasma with HIV-1 and HTLV-Iin human T lymphocytes. AIDS Res. Hum. Retroviruses 8: 1863-1868. Schaeverbeke, T., Gilroy, C.B., Bebear, C., Dehais, J., and

702

28)

29)

30) 31) 32)

K. MATSUDA

Taylor-Robinson, D. 1996. 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: 824-828. Schaeverbeke, T., Vernhes, J.P., Lequen, L., Bannwarth, B., Bebear, C., and Dehais, J. 1997. Mycoplasmas and arthritides. Rev. Rhum. Engl. Ed. 64: 120-128. Schaeverbeke, T., Renaudin, H., Clerc, M., Lequen, L., Vernhes, J.P., De Barbeyrac, B., Bannwarth, B., Bebear, C., and Dehais, J. 1997. Systematic detection of mycoplasmasby culture and polymerase chain reaction (PCR) procedures in 209 synovial fluid samples. Br. J. Rheumatol. 36: 310-314. Svennerholm, L. 1956. The quantitative estimation of cerebrosides in nervous tissue. J. Neurochem. 1: 42. Svennerholm, L. 1963. Chromatographic separation of human brain gangliosides. J. Neurochem. 10: 613-623. Tsai, S., Wear,D.J., Shih, J.W.,and Lo, S.C. 1995. Mycoplasmas and oncogenesis: persistent infection and multistage malignant transformation. Proc. Natl. Acad. Sci. U.S.A. 92:

ET AL

10197-10201. 33) Weidenfeld, J., Wohlman, A., and Gallily, R. 1995. Mycoplasma fermentans activates the hypothalamo-pituitary adrenal axis in the rat. Neuroreport 6: 910-912. 34) Williams, M.H., Brostoff, J., and Roitt, I.M. 1970. Possible role of Mycoplasma fermentans in pathogenesis of rheumatoid arthritis. Lancet ii: 277-280. 35) Williams, M.H., and Bruckner, F.E. 1971. Immunological reactivity of Mycoplasma fermentans in patients with rheumatoid arthritis. Ann. Rheum. Dis. 30: 271-273. 36) Yirmiya, R., Barak, O., Avitsur, R., Gallily, R., and Weidenfeld, J. 1997. Intracerebral administration of Mvcoplasma fermentans produces sickness behavior: role of prostaglandins. Brain Res. 749: 71-81. 37) Zhang, B., Shih, J.W., Wear, D.J., Tsai, S., and Lo, S.C. 1997. High-level expression of H-ras and c-myc oncogenes in mycoplasma-mediated malignant cell transformation. Proc. Soc. Exp. Biol. Med. 214: 359-366.