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*
Max-Planck Institute for Infection Biology, Department of Immunology, Berlin, Germany;
Free University-Berlin, Department of Biology, Chemistry, and Pharmacy, Berlin, Germany; and
Department of Microbiology, Colorado State University, Fort Collins, CO 80523
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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). This capability has been
attributed in part to their cell wall, which consists of various unique
lipids. Some of these are important in the host-pathogen interaction,
such as resistance against microbicidal effector mechanisms and
modulation of host cell functions, and/or are presented as Ags to T
cells. Here we show that two lipids are released from the mycobacterial
cell wall within the phagosome of infected M and transported out of
this compartment into intracellular vesicles. One of these lipids was
identified as lysocardiolipin. Lysocardiolipin was generated through
cleavage of mycobacterial cardiolipin by a Ca2+-independent
phospholipase A2 present in M lysosomes. This result
indicates that lysosomal host cell enzymes can interact with released
mycobacterial lipids to generate new products with a different
intracellular distribution. This represents a novel pathway for the
modification of bacterial lipid Ags. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)3 as well as the
activation of lymphocytes (4, 5). Lipoarabinomannan (LAM)
suppresses immune responses by various mechanisms including impaired T
cell activation, inhibition of IFN-
-mediated M activation,
scavenging of free oxygen radicals, and inhibition of protein kinase C
activity (4, 6). Phosphatidylinositolmannosides (PIMs)
inhibit Ag-specific proliferation of peripheral blood monocytes
(7). Phthiocerol dimycocerosate and its functional
localization in the mycobacterial cell wall were shown to be crucial
for survival of Mycobacterium tuberculosis in the lungs of
mice (8). Mycobacterial phospholipids have also been
implicated in immunomodulation; human blood monocytes infected with
M. tuberculosis release phosphatidylethanolamine and
phosphatidylinositol of mycobacterial origin, which activate suppressor
lymphocytes (9). Lipids of mycobacteria can be recognized
by the immune system as Ags. In humans, T cells specific for
mycobacterial glycolipids are considered part of the immune response
against M. tuberculosis (10). Mycobacterial
lipids of various complexities, such as LAM,
PIMX, mycolic acid, glucose monomycolate, and
isoprenoid glycolipids, are presented to T cells by CD1 molecules
leading to lysis of infected Ms and killing of M.
tuberculosis (11, 12). It is not known yet whether
mycobacterial lipids need to be processed by host cells to become
antigenic although it was recently reported that galactosylceramide
derivatives require an enzymatic cleavage before they are recognized by
NK T cells in the context of CD1d (13). Our experiments
show that mycobacterial lipids are released from mycobacteria,
transported out of the phagosome, and altered by host cell enzymes. In
the case of cardiolipin, its cleavage by a lysosomal-type
Ca2+-independent phospholipase
A2 occurs before the transfer of lysocardiolipin
out of the phagosome. We postulate that this processing pathway can
generate biologically active lipid derivatives that play a role during
infection by and persistence of mycobacteria within their
host. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All reagents were purchased from Sigma (Deisenhofen, Germany), if not indicated otherwise.
Cell culture
M from 6- to 8-wk-old C57BL/6 mice were differentiated in
tissue culture medium containing 20% L929 cell supernatant (American
Type Culture Collection, Manassas, VA). To label M lipids
radioactively, 107 M were incubated for
12 h in the presence of 10 µCi
[14C]palmitic acid, 50 Ci/mmol (Hartmann,
Braunschweig, Germany).
Bacteria and infection
M. bovis bacillus Calmette-Guérin (BCG;
Copenhagen strain) was cultured in 7H9 Middlebrook medium (Difco,
Detroit, MI) supplemented with albumin dextrose catalase
(Difco), and harvested during logarithmic growth phase. Bacterial
lipids were radioactively labeled by incubating
108 M. bovis BCG for 12 h in the
presence of 10 µCi [14C]palmitic acid. M
were infected at a 10:1 multiplicity of infection.
Subcellular fractionation
Mycobacteria containing phagosomes were prepared using a 12/50% sucrose step gradient as previously described (14). The top layer, containing intracellular vesicles, and the 12/50% interface consisting predominantly of mycobacterial phagosomes were collected. The supernatant was diluted 1/10 with PBS and centrifuged for 20 min at 3200 x g to remove any remaining bacteria. To recover all intracellular vesicles, the supernatant was further centrifuged at 100,000 x g for 1 h. No bacteria were observed in this preparation (data not shown). The 12/50% interface was washed once in PBS, and the mycobacteria were stripped off the phagosomal membranes by several Nonidet P-40 washes. The resulting pellet contained the mycobacteria (data not shown). The supernatants from the washes contained the phagosomal membrane and were combined and lyophilized.
To obtain lysosomes, noninfected M were lysed and the lysate was
layered on top of a 20% Percoll gradient (Biochrom, Berlin, Germany)
in triethanolamine buffer (pH 6.5) and centrifuged for 21 min at
25,000 x g. The lysosome-enriched fractions were
separated from the Percoll by centrifugation for 1 h at
100,000 x g. These fractions were highly enriched in
enzyme activity per milligram of protein for the lysosomal marker
enzyme 
-galactosidase, and the proteins lysosome-associated membrane
protein-1 and cathepsin D as determined by Western blot (data
not shown). For cleavage experiments, lysosomes derived from 5 x
106 M were disrupted in 1% Triton X-100, 50
mM acetate, pH 4, before the addition of purified mycobacterial lipids.
Mixtures were incubated at 37°C for 2 h. Reaction was stopped by
freezing, and samples were lyophilized, extracted, and analyzed by high
performance thin layer chromatography (HPTLC).
Lipid extraction and HPTLC analysis
Lipids were extracted overnight in chloroform/methanol (2:1;
v/v). Samples were spotted in one corner of a 10 x 10 cm HPTLC
plate (Merck, Darmstadt, Germany). The HPTLC plate was developed in the
first dimension in chloroform/methanol/ammonium hydroxide (80:20:2;
v/v/v) to 8 cm, and, in the second dimension, in propanol/water/acetic
acid (80:10:10; v/v/v) to 8 cm. If one-dimensional analysis was
performed, chloroform/methanol/water (65:25:4; v/v/v) was used as
solvent system. Lipids were analyzed using the following spray
reagents: 0.2% ninhydrin in ethanol, a phosphorus dip (Dittmers
reagent), and 1% 
-naphthol in 5%
H2SO4/ethanol
(15). Radioactive lipids were visualized and quantified
using a phosphoimager (Fujix BAS 1000; FUJIX, Tokyo, Japan). For
the analysis of single lipids, the radioactive lipids were scraped off
the HPTLC plate and re-extracted, and radioactivity was measured.
Purification of lipids
Lipids from 20 g lyophilized M. bovis BCG were extracted as described above. The total lipid extract was chromatographed by multiple silica gel chromatography, applying chloroform/methanol gradients in 10% steps (16). The fraction containing the highest concentration of lipid 4, which is cleaved by lysosomal enzymes, was further separated on C18 Sep-Pak cartridge (Waters; Millipore, Bedford, MA). In the last step this lipid was purified by preparative HPTLC.
Lipid identification
To identify fatty acids, 200 µg lipid was dried and methanolyzed as described (17). The resulting methylesters were analyzed by gas chromatography-mass spectrometry (GC-MS; Hewlett-Packard, Palo Alto, CA). The sample was analyzed by 1H- and 13C-nuclear magnetic resonance (NMR) (300 MHz, Varian-300 NMR; Varian Associates, Palo Alto, CA), matrix-associated laser desorbtion ionization time of flight mass spectrometry (MALDI-MS) (Voyager Elite; PerSeptive Diagnostics, Cambridge, MA; 2, 5 dihydroxybenzoic acid was used as matrix), and electron spray ionization mass spectrometry (ESI-MS) (V6 Quattro-SQ; Fisons Instruments, Loughborough, U.K.).
Incubation of mycobacterial cardiolipin with phospholipase A2
14C-labeled cardiolipin was lyophilized and resuspended in 0.1% Triton X-100 in PBS (pH 8.9) followed by the addition of 10 U phospholipase A2 (porcine pancreas), and the mixture incubated at 25°C for 1 h. The lipids were extracted and analyzed by HPTLC.
Phospholipase A2 characterization
Lysosomes of 5 x 106 M were
disrupted in 0.1% Triton X-100, 5 mM EGTA (in case of
Ca2+ addition, EGTA was exchanged with 10 mM
CaCl2). Buffers varying in their pH contained 50
mM acetate (pH 4, pH 6.2), or 50 mM Tris (pH 7, pH 8.5). The effects of
the two phospholipase A2 inhibitors,
p-bromophenacyl bromide (pBPB) and diethyl
p-nitrophenyl phosphate (DENP), were determined after
preincubation for 30 min at 37°C as described (18). The
phospholipase A2 substrate 5 µCi
choline-methyl-[3H]phosphatidylcholine was
added, and mixtures were sonicated and incubated at 37°C for 2
h. Reaction was stopped and samples were separated by HPTLC.
Radioactivity was quantified with a phosphoimager, and the amount of
the cleavage product lysophosphatidylcholine was expressed as a
percentage of maximal cleavage observed at pH 4 in the absence of
Ca2+.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, cardiolipin is released from the bacteria and
subsequently cleaved by a lysosomal phospholipase
A2. The resulting lysocardiolipin could be
detected within intracellular vesicles distinct from phagosomes.
Mycobacterial lipid content of different cellular
compartments of infected M
M infected with [14C]palmitic
acid-labeled mycobacteria were lysed at different time points after
infection (2, 8, and 48 h), fractionated into mycobacteria,
phagosomal membranes, and other intracellular vesicles, and analyzed
for their lipid content by HPTLC (Fig. 1
). No obvious alterations were observed
in the lipid pattern of the mycobacterial fraction during the time
course of the experiment, with one notable exception. Lipid 1 was not
detectable in the 2-h pattern but appeared after 8 h and was even
more abundant after 48 h (retardation factor
(Rf): 0.21/0.85; Fig. 1). In addition, the
amount of trehalosedimycolate (TDM) present in the mycobacterial
fraction increased by 47% between 2 and 48 h postinfection
(p.i.) when compared with the total radioactivity of the lipids
in this fraction. The lipid patterns of the phagosomal membrane
fraction and the vesicle fraction differed from those seen in the
mycobacterial fraction. For both fractions, the number of different
lipid species, as well as the total amount of radioactivity, increased.
Thus, the total radioactivity recovered from the vesicle fraction at
48 h postinfection was 14 times higher than the one measured at
2 h.
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Lipid patterns of radioactively labeled M and M.
bovis BCG were compared with those of vesicles of M infected
with [14C]palmitic acid-labeled M.
bovis BCG for 48 h and the respective M. bovis BCG
isolated (Fig. 2). The lipid pattern of
M. bovis BCG was much more complex than that of the M,
which consisted of 17 major lipids compared with >30 in the case of
M. bovis BCG. Comparison between M. bovis BCG
grown in culture (B) and intracellularly (C)
revealed that the relative amount of different lipid species varied
between these two conditions, e.g., the amount of TDM was higher in
intracellularly grown mycobacteria. The pattern of the vesicles of the
infected M roughly resembled that of uninfected M, indicating
that either mycobacterial lipids were metabolized by M, or dominant
lipid compounds transported out of the phagosome were identical in both
M and mycobacteria. Two lipids (3a and 2) present in the vesicle
fraction were not detectable in uninfected M, indicating that they
were specific for mycobacteria (Rf 3a: 0.17/0.86;
2: 0.73/0.98). Lipid 2 is probably TDM-related and currently analyzed
in detail. Lipid 3a was not identical with lipid 1 as revealed by
different Rf values (Fig. 2) and comparative
HPTLC (data not shown). Furthermore, lipid 3a was not present in the
M. bovis BCG pattern; therefore, it represents a cleavage
product modified after its release from the bacteria and before its
transport to other vesicles.
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can modify mycobacterial lipids
To study processing, radioactively labeled mycobacterial lipids
were isolated by HPTLC and incubated with purified M lysosomes.
Although most of the lipids were not affected by this treatment (data
not shown), lipid 4 was cleaved, resulting in two new products (Fig. 3), termed 3b and 5. Thus, lysosomal M
enzymes can modify mycobacterial lipids into distinct products.
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The characteristics of lipid 4 were examined using three different
spray reagents revealing that it did not contain amino or carbohydrate
groups, but did contain phosphorous (Fig. 4a). After methanolyzing, the
lipid was analyzed by GC-MS to define its fatty acid content (Fig. 4b). The two major fatty acids were identified as
hexadecanoic acid (C16) and octadecenoic acid (C18:1).
1H-NMR analysis confirmed that one of the fatty
acids was unsaturated (5005 ppm), whereas the characteristic signals of
carbohydrates or carbonyls were not detected (data not shown). The
ESI-MS spectrum confirmed a high purity of the product (>99%) because
no mass peaks were observed between 350 and 650 m/z (data
not shown). The major peak at 701 m/z was double-nosed,
suggesting that the analyzed lipid is double-negatively charged and
probably has a molecular mass of 1402 Da (M2+).
MALDI-MS analysis verified the molecular mass
(M+) of 1403 Da (Fig. 4c). From the
combined data obtained we propose that the lipid represents a
phospholipid with C16 and C18:1 fatty acids and a molecular mass of
1404 Da. The only known mycobacterial phospholipid with such
properties is diphosphatidylglycerol (cardiolipin), which is one of the
major mycobacterial phospholipids (19). The identification
was confirmed by comparative HPTLC using a cardiolipin standard derived
from bovine heart revealing identical Rf values
(Fig. 4d). Analysis of the standard cardiolipin by ESI-MS
gave a similar double-nosed peak, but at 723 Da, which is due to the
different fatty acid content and higher unsaturation of the fatty acids
of cardiolipin derived from heart (data not shown).
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To see whether cardiolipin is released from mycobacteria,
radioactively labeled M. bovis BCG were cultured in
Middlebrook medium for 24 h, and the culture supernatant (CSN) was
collected, lyophilized, extracted, and analyzed by HPTLC. Although the
HPTLC pattern of CSN lipids reveals a number of lipid species also
present in the cellular pattern, only certain lipids are released into
the CSN, suggesting that this represents rather a selective process and
is not just an effect of cell lysis (data not shown). Among these
lipids a significant amount of cardiolipin is detected in the CSN (Fig. 4
e).
Cardiolipin is hydrolyzed by lysosomal-type Ca2+-independent phospholipase A2
Radioactively labeled mycobacterial cardiolipin was incubated in
vitro with various phospholipases. Only in the case of phospholipase
A2 the resulting cleavage products were identical
with the products derived by incubation with lysosomes (Fig. 5a). Comparison of the
migration properties of the cleavage products with known cardiolipin
catabolites and HPTLC analysis with the respective standards indicate
that lipid 3c was identical with lysocardiolipin and lipid 5 to
di-lysocardiolipin (data not shown). Lipid 3a from a vesicle
preparation, lipid 3b from incubation of cardiolipin with lysosomes,
and lipid 3c from incubation of cardiolipin with phospholipase
A2 were re-extracted and developed on a HPTLC.
Lipid 3a had an Rf value identical with the
products of cardiolipin cleaved by either purified lysosomes or
phospholipase A2 (Fig. 5b). This shows
that M lysosomes contain a phospholipase A2
activity that can cleave mycobacterial cardiolipin released from
intracellular bacteria into lysocardiolipin and di-lysocardiolipin. To
characterize the type of phospholipase activity present in M
lysosomes, the phospholipase A2 substrate
[3H]-labeled phosphatidylcholine was incubated
with purified lysosomes under various conditions (Fig. 6). The activity of the lysosomal
phospholipase A2 was calcium-independent because
depletion of calcium with EGTA had no effect. The phospholipase
A2 activity was optimal at pH 4, reduced by 66%
at pH 6.2, and totally abolished at pH 7 and pH 8.5, indicating the
requirement of an acidic milieu for enzymatic activity. The
phospholipase A2 inhibitor pBPB blocked the
activity by 32%, whereas DENP completely suppressed the enzymatic
activity. DENP is a serine protease inhibitor recently described
to block lysosomal-type Ca2+-independent
phospholipase A2 isolated from pneumocytes
(18).
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express a lysosomal-type
Ca2+-independent phospholipase
A2 that can cleave mycobacterial cardiolipin
allowing subsequent detection of lysocardiolipin outside of the
phagosome. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In earlier studies from our laboratory, confocal microscopy of infected
murine M revealed that large quantities of mycobacterial cell wall
material are transferred from the phagosome and accumulate in late
endosomes/lysosomes. LAM and phosphatidylinositoltetramannoside
(PIM4) have been determined as the main
transfered lipids (20). Furthermore, lipids including
cardiolipin are also released in a selective way from in vitro grown
mycobacteria into the CSN. Here we demonstrate by using radioactively
labeled mycobacteria that apolar lipids are also released from
intracellular mycobacteria into the phagosomal lumen and traffic out of
the phagosome into other intracellular compartments, which is in
accordance to two recent studies (20, 21). This indicates
that mycobacterial lipids with diverse chemical properties are
transferred out of the phagosome. The kinetics of the appearance of
different lipid species in various subcellular fractions of infected
M revealed several interesting results. When compared with the total
radioactivity of the lipids extracted from the mycobacterial fraction,
the amount of radioactive TDM in the mycobacteria increased by 47%
between 2 and 48 h p.i. TDM has been implicated in interfering
with phagosome-lysosome fusion; therefore, it can be speculated that
within cells, production of this glycolipid is up-regulated to prevent
fusion of the phagosome with lysosomes (22). The amount of
radioactivity measured in the total lipid extract of the vesicle
fraction increased 14-fold between 2 and 48 h p.i. This indicates
that mycobacterial lipids are transported out of the phagosome into
other cellular compartments in a time-dependent manner. Comparison of
lipid patterns of mycobacteria and noninfected M revealed that a
number of the radioactively labeled lipids that were present in
nonphagosomal vesicles had the same Rf values as
M lipids. We assume that transferred mycobacterial lipids either
have the same Rf values as eukaryotic lipids
and/or are metabolically degraded, permitting the transfer of
radioactivity to M lipids. By using purified lipids, we could
demonstrate that intact M can degrade major mycobacterial lipids
such as PIM4 and mycoside B, and that this leads
to the integration of radioactivity into M lipids such as
phosphatidylinositol, phosphatidylethanolamine, and
phosphatidylglycerol (data not shown). This observation is in contrast
to earlier statements claiming that mycobacterial lipids are highly
resistant to degradation by the host cell (23).
We have shown that mycobacterial lipids are transported to late endosomes/lysosomes containing several glycolipid-degrading enzymes such as lipases and glycosidases. However, the use of purified lysosomes and individual lipids revealed that lysosomal enzymes did not alter the majority of the mycobacterial lipids investigated. In contrast, cardiolipin, an abundant and integral compound of the mycobacterial envelope, was cleaved by a lysosomal phospholipase A2 into lysocardiolipin and di-lysocardiolipin. Cardiolipin is present in the inner membrane of prokaryotes as well as in mitochondria of eukaryotic cells, most prominently in the heart muscle of mammals (24). Cardiolipin itself was associated with the mycobacteria during the entire infection period of 48 h, whereas lysocardiolipin was transferred out of the phagosome into other vesicles, indicating that cleavage of cardiolipin occurred in the phagosome. Interestingly, cardiolipin was released from in vitro grown mycobacteria, suggesting that within the phagosome, released cardiolipin is cleaved immediately. The phospholipase activity responsible for the cleavage is of host cell origin, because mycobacteria do not express surface-exposed phospholipase A (25). Furthermore, lysocardiolipin was not found in the CSN. A lysosomal-type Ca2+-independent phospholipase A2 is responsible for the degradation of mycobacterial cardiolipin, which is distinct from the groups of the secreted or cytoplasmic phospholipases A2. Evidence that this type of phospholipase interacts with mycobacterial lipids indicates a new function for these lysosomal enzymes, which could be important during mycobacterial infection. Enzyme activity was detected at pH 6.2, which is the pH value of intact mycobacterial phagosomes, but optimal activity was observed at low pH (26). This indicates that the cleavage could occur at a pH of 6.2 in the phagosome or more effectively in the phagolysosome with its lower pH. Acidified phagolysosomes contain most probably nonviable bacteria (14). One possible function for this phospholipase could be processing of potential lipid Ags, such as the B cell Ag cardiolipin, suggesting a similar role as lysosomal proteases in Ag processing for MHC class II presentation. Moreover, host cells up-regulate their lysosomal phospholipase A activity following mycobacterial infection (27). This may hint toward a bactericidal activity of this enzyme as shown for other phospholipase A2 types (28). It may also be a direct response to the increased amount of phospholipids in the phagosome, facilitating degradation of cardiolipin, and release of lysocardiolipin. The presence of lysophospholipids, namely lysocardiolipin, could have important effects on the host cell.
It has been shown that lysophospholipids can suppress IL-2 secretion of
activated CD4 T cells and inhibit membrane fusion
(29, 30, 31). Pathogenic mycobacteria survive in M and
reside within phagosomes that do not fuse with lysosomes
(26). Therefore, lysocardiolipin may be a candidate lipid
involved in preventing phagolysosome fusion or suppression of CD4 T
cell functions (26, 31). Furthermore, lysophospholipids
can influence signal transduction pathways, e.g., by stimulating
arachidonic acid release and activating protein kinase C (32, 33).
The ability of M to modify mycobacterial cardiolipin, which
subsequently leads to an altered intracellular distribution of
lysocardiolipin, could be crucial for the biological
effects of cardiolipin. We envisage this novel processing
pathway as a general mechanism of how infected cells alter lipids
released from intracellular pathogens. The resulting lysophospholipids
are likely to have functions that influence the outcome of
mycobacterial infections.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ulrich E. Schaible, Department of Immunology, Max-Planck Institute for Infection Biology, Schumannstraße 20/21, 10117 Berlin, Germany. E-mail address: schaible{at}mpiib-berlin.mpg.de
3 Abbreviations used in this paper: M, macrophage; LAM, lipoarabinomannan; PIM, phosphatidylinositolmannoside; PIM4, phosphatidylinositoltetramannoside; p.i., postinfection; BCG, bacillus Calmette-Guérin; HPTLC, high performance thin layer chromatography; GC-MS, gas chromatography-mass spectrometry; NMR, nuclear magnetic resonance; MALDI-MS, matrix associated laser desorbtion ionization time of flight mass spectrometry; ESI-MS, electron spray ionization mass spectrometry; pBPB, p-bromophenacyl bromide; DENP, diethyl p-nitrophenyl phosphate; TDM, trehalosedimycolate; CSN, culture supernatant; Rf, retardation factor.
Received for publication March 19, 2001. Accepted for publication June 12, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,-trehalose 6,6'-dimycolate) inhibits fusion between phospholipid vesicles. Proc. Natl. Acad. Sci. USA 88:737.This article has been cited by other articles:
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  O. H. Vandal, M. H. Gelb, S. Ehrt, and C. F. Nathan Cytosolic Phospholipase A2 Enzymes Are Not Required by Mouse Bone Marrow-Derived Macrophages for the Control of Mycobacterium tuberculosis In Vitro Infect. Immun., March 1, 2006; 74(3): 1751 - 1756. [Abstract] [Full Text] [PDF]
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P. Domenech, M. B. Reed, and C. E. Barry III Contribution of the Mycobacterium tuberculosis MmpL Protein Family to Virulence and Drug Resistance Infect. Immun., June 1, 2005; 73(6): 3492 - 3501. [Abstract] [Full Text] [PDF]
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 K. Fischer, E. Scotet, M. Niemeyer, H. Koebernick, J. Zerrahn, S. Maillet, R. Hurwitz, M. Kursar, M. Bonneville, S. H. E. Kaufmann, et al. Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1d-restricted T cells PNAS, July 20, 2004; 101(29): 10685 - 10690. [Abstract] [Full Text] [PDF]
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 S. H.E. Kaufmann and U. E. Schaible A Dangerous Liaison between Two Major Killers: Mycobacterium tuberculosis and HIV Target Dendritic Cells through DC-SIGN J. Exp. Med., January 6, 2003; 197(1): 1 - 5. [Full Text] [PDF]
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 D. G. Russell, H. C. Mwandumba, and E. E. Rhoades Mycobacterium and the coat of many lipids J. Cell Biol., August 5, 2002; 158(3): 421 - 426. [Abstract] [Full Text] [PDF]
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 K. Fischer, H. Collins, M. Taniguchi, S. H. E. Kaufmann, and U. E. Schaible IL-4 and T Cells Are Required for the Generation of IgG1 Isotype Antibodies Against Cardiolipin J. Immunol., March 15, 2002; 168(6): 2689 - 2694. [Abstract] [Full Text] [PDF]
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