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Production in Response to Live or Dead Bacteria by TNF and Other Factors1
Department of Microbiology, University of Melbourne, Parkville, Victoria, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production. In addition to being poor inducers of IL-12, killed
bacteria actively suppressed IL-12 production in response to live
bacteria, by mechanism(s) unknown. The implications of these findings
are discussed in light of the fact that only live bacteria
satisfactorily induce cell-mediated immunity to
infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, which in turn activates
macrophage bactericidal mechanisms (1), while CD8+ T cells
lyse infected cells in a perforin-dependent (2), IFN--independent
manner (3). Protection against intracellular organisms is generally
satisfactorily induced only by infection with live persistent
organisms. Although repeated injection of killed L.
monocytogenes into CD4-deficient mice induced protective
CD8+ T cells (4), injections of killed bacteria into intact
mice are generally not protective (1, 5-9) and may favor instead IL-4
production by the T cells (10). These observations have profound
implications for vaccine development. The quest for improved vaccines
with fewer side effects to diseases such as tuberculosis and leprosy
may well depend on our understanding of how the response to protein
vaccines might be biased toward the induction of IFN--producing T
cells.
One of the early events in infection with intracellular bacteria is
phagocytosis by resident macrophages and the release of cytokines by
these cells. A key role is played by IL-12, which up-regulates the
production of IFN- by T cells and NK cells (11). Depletion of IL-12
decreases IFN- production and exacerbates infection in a number of
intracellular infections (11). Injection of killed Listeria
together with recombinant IL-12 leads to IFN- production and
protective ACR, not induced by killed Listeria alone (9).
But what regulates the induction of IL-12? In vivo only live
Listeria organisms induce IL-12 production (12), but in
vitro observations have been conflicting. Heat-killed
Listeria (13, 14) or heat-killed mycobacteria (15) or even
phagocytosis of latex beads (16, 17) induced IL-12 production by mouse
peritoneal macrophages or human monocytes. Song et al. (18) suggested
that mouse splenic adherent macrophages were triggered to produce IL-12
by both live and killed Listeria but that whole spleen cell
cultures in the presence of killed Listeria produced IL-10
which suppressed IL-12.
With these contradictions in mind, we investigated the control of IL-12 production in response to two murine model infections. L. monocytogenes causes an acute infection which has been widely studied as a model of ACR (1). Infection of mice with the vaccine strain 19 of Brucella abortus, on the other hand, causes a chronic infection in which IL-12 also plays a crucial role in the induction of ACR (19). We have previously shown that live Listeria and live Brucella organisms are much more efficient than heat-killed organisms at inducing macrophages in vitro to produce TNF (20). The present experiments with TNF receptor knockout mice indicate that TNF plays an important role in enhancing IL-12 production in vitro and in vivo. Furthermore, killed bacteria appear to actively down-regulate IL-12 production in vivo, although injection of mAb to IL-10 suggested this cytokine was not involved in down-regulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/10 mice were bred by pedigreed brother-sister mating in the Department of Microbiology and Immunology, University of Melbourne (Melbourne, Australia). They were housed under conditions of isolation and fed sterile pellets and water to maintain their infection-free status. Gene-targeted knockout mice lacking both the p55 and p75 receptors for TNF were produced by J. Peschon (21) and used with his kind permission. They were bred by Dr. Janet Ruby of the Department of Microbiology and Immunology, University of Melbourne (Melbourne, Australia).
Bacteria and antigens
L. monocytogenes and B. abortus strain 19 were maintained by weekly subculture on horse blood agar (HBA). Cultures were periodically renewed from freeze-dried stock to maintain a constant level of virulence for the mice. Heat-killed Listeria (HKL) and heat-killed Brucella (HKB) were prepared by heating a viable bacterial suspension of known concentration for 60 min at 70°C. Viability was checked by sampling onto HBA. For infection and immunization, C57BL/10 were injected i.v. with L. monocytogenes or B. abortus in 0.2 ml of normal saline or with heat-killed bacteria in the same volume.
Quantitation of infection
Mice were killed by CO2 narcosis. Spleen and liver were weighed. Weighed fragments of spleen and liver were homogenized in 5 ml 0.1 M PBS, pH 7.2 with an Ultra Turrax homogenizer (Janke and Kunkel, Breisgau, Germany). The numbers of L. monocytogenes or B. abortus in the organs were established by plating serial dilutions of homogenates on HBA.
Preparation of serum and spleen homogenate supernatants
Mice were bled from the heart under fluothane anesthesia. Individual sera were isolated from clotted blood by centrifugation. Spleen homogenate supernatants were prepared by centrifuging at 2000 x g for 7 min the spleen homogenates prepared for bacterial counts above.
Cytokine and mAbs to cytokines
Recombinant murine IL-12 (2.3 x 108 U/mg
protein) was kindly provided by Dr. Maurice Gately (Hoffman la Roche,
Nutley, NJ). Recombinant murine IL-10 was purchased from Endogen
(Woburn, MA). Anti-IL-12 mAb was prepared from hybridoma C15.6 (rat
IgG1, kindly provided by Dr. G. Trinchieri, Wistar Institute,
Philadelphia, PA). MAb to IL-4 was prepared from hybridoma 11B11 (22),
mAb to IL-10 from hybridoma SXC-1 and SXC-4 (23), mAb to TNF from XT22
(24), and MAb to IFN- from hybridoma HB170 (25). All mAbs were used
as ammonium sulfate-precipitated protein from ascitic fluid.
Cell culture for cytokine production
Resident peritoneal cells were collected by lavage with 5 ml of
DMEM with 10% FCS (DMEM + FCS) containing 10 U/ml heparin. Cells were
centrifuged through an FCS cushion and resuspended in DMEM + FCS.
Spleens were pooled within experimental groups and teased through an 80
mesh stainless steel sieve into DMEM + FCS. The spleen cell suspension
was centrifuged at 800 x g for 7 min, suspended in
Tris-buffered 0.83% ammonium chloride to lyse erythrocytes, underlaid
with 1 ml of FCS to remove debris, and centrifuged again. The cells
were resuspended in DMEM + FCS. Resident peritoneal cells were cultured
at a concentration of 2 x 106 per well, while spleen
cells were cultured at 4 x 106 cells per well in the
presence or absence of indicated Ags in 2-ml volumes. In experiments
using live bacteria as in vitro stimulus, the medium was prepared
without antibiotics. After 24 h supernatants were harvested,
filtered, and assayed for IL-12 or IFN-.
Bioassay for murine IL-12
This Ab capture assay (26) relies on the ability of IL-12 to
promote the production of IFN- by normal spleen cells in the
presence of suboptimal concentrations of IL-2. Flat-bottom 96-well
plates (Nunc, Roskilde, Denmark) were coated at 4°C overnight with
sterile anti-IL-12 mAb C15.6 at a concentration of 20 µg/ml in
0.1 M sodium carbonate-bicarbonate buffer (pH 9.5). The plates were
washed three times with PBS, and remaining binding sites were blocked
with 5% FCS in PBS for 1 h at 37°C and washed again. Triplicate
100-µl samples of serially diluted culture supernatants were added to
some wells. Serially diluted rIL-12 for a standard curve and medium as
background were added to other wells. The plates were incubated for 4
to 5 h at room temperature and then washed with PBS. To all
plates, 100 µl/well containing BALB/c splenocytes 5 x
106/ml and 50 U/ml recombinant murine IL-2 were added. The
cells were then cultured for 24 h, and 50 µl of supernatants
were transferred to another set of 96-well flat-bottom plates for
IFN- assay (see below). IL-12 concentration was calculated against
the standard curve obtained with serial dilutions of rIL-12. The limit
of detection of bioactive IL-12 by this assay is 10 pg/ml, but in some
experiments the samples were diluted initially, giving a higher limit.
IFN- bioassays
IFN- titers were determined by comparing the suppression of
the proliferation of WEHI-279 cells (27) with an IFN- standard
supplied by the National Institutes of Health (Bethesda, MD). The
specificity of IFN- assay was checked by incorporating
anti-IFN- mAb into replicate wells. Neutralization of 90% or
more of the activity was accepted as indicating specificity.
NO2- assay
Nitric oxide production by spleen cells was determined by the Griess reaction (28). Culture supernatants (50 µl) were mixed with 100 µl of 1% sulfanilamide (Sigma, St. Louis, MO) and 100 µl of 0.1% N-1-naphthylethylenediamine dihydrochloride in 2.5% polyphosphoric acid (Sigma) at room temperature. Absorbance at 540 nm was measured. NO2- was quantified by comparison with Na(NO2) (Sigma) as a standard.
Statistical analysis
The statistical significance of data was determined by Student’s t test.
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Results |
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Since only infection with live, virulent organisms induces
acquired cellular resistance in vivo and since IL-12 is apparently the
key cytokine controlling activation of IFN--producing T cells, the
induction of IL-12 in response to live or killed bacteria was
investigated in vivo and in vitro. Mice were injected i.v. with 2
x 107, 2 x 108, or 2 x
109 heat-killed Listeria organisms or with
2 x 104 or 2 x 106 live
Listeria. Groups of mice were killed at intervals, and serum
was collected for bioassay of IL-12, while bacterial counts were
performed on spleen homogenates. Results are shown in Figure 1
. A sharp peak of IL-12 in the serum was
observed 24 h after injection of live bacteria. In mice receiving
2 x 104 live Listeria, bacterial numbers
continued to increase after 24 h, but IL-12 in the serum fell to
negligible titers. Mice receiving 2 x 106 Listeria did not survive longer than 24 h, but
the high numbers of bacteria induced even higher titers of IL-12.
Injection of heat-killed Listeria in doses ranging from
2 x 107 to 2 x 109 failed to induce
IL-12 production. In a similar experiment with B. abortus,
mice were injected with 5 x 105 or 5 x
107 live organisms or with 4 x 109
heat-killed organisms. The results in Table I show a longer time scale in production
of IL-12, particularly in response to the higher dose of B.
abortus. Nevertheless, production terminated before the
elimination of bacteria. Again, killed bacteria failed to induce the
production of IL-12 detectable in the serum.
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).
Graded doses of the organisms were necessary for comparison because
live Listeria increased from an inoculum as low as 10
organisms to >109. Live Listeria induced more
IL-12 from either peritoneal or spleen cells than did heat-killed
Listeria. Production of IL-12 was enhanced by activation of
the macrophages by prior infection. There was a tendency, more marked
among the wells containing killed bacteria, for high numbers of
bacteria to suppress production of IL-12.
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Since we had shown previously that live bacteria, either
Listeria or Brucella, induced more TNF from
peritoneal cells in vitro than did heat-killed bacteria (20), the role
of TNF in production of IL-12 in vitro and in vivo was investigated
using TNF receptor knockout mice. The response of spleens cells from
TNFR-/- or wild-type mice was tested in vitro by the addition of live
Listeria or Brucella organisms. IL-12 in the
supernatants was assayed 24 h later (Table III). It was notable that
Listeria was a stronger stimulus than Brucella.
Nevertheless, whereas both Listeria and Brucella
elicited the production of IL-12 from cells of wild-type mice,
negligible amounts of IL-12 were produced by the cells that lacked a
TNF receptor.
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). At
this stage, the infection was not greatly exacerbated in the TNFR-/-
mice, but IL-12 production in these mice lacking a TNF response was
negligible. Uninfected mice showed 0.01 ng/ml IL-12 in their serum.
This experiment was confirmed by depleting TNF by injecting 2 mg of mAb
i.p. 3 and 1 day before infection with 2 x 104 Listeria. In this case, IL-12 levels in the infected
mice decreased from 1.1 ± 0.6 ng/ml to 0.3 ± 0.2 ng/ml
(p < 0.05). Thus, acute depletion of TNF had a
similar effect to the chronic inability to respond to TNF seen in
TNFR-/- mice.
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). Two weeks after
infection, the difference in bacterial counts between TNFR-/- and
wild-type mice was >10-fold, but immunopathology, reflected in spleen
weight, was less in the TNFR-/- mice (Table V). IFN- production by lymphocytes
cultured in the presence of specific Ag was unaffected by loss of TNF
responsiveness, but NO production was diminished. NO production did not
require the induction of IFN- in these cultures, being as high in
cultures stimulated nonspecifically by heat-killed Listeria
as in the specifically stimulated cultures.
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In Listeria infection, IL-12 production was clearly
down-regulated after 24 h of infection, despite continuing high
numbers of bacteria, as in Figure 1
. On the hypothesis that this might
be due to late induction of antiinflammatory cytokines, we tested the
ability of mAb directed against candidate cytokines to enhance or
prolong IL-12 production. This required the prior testing of the
effectiveness of the mAb in vivo. The most likely candidate for
down-regulation of IL-12 production is IL-10 (11, 18). IL-4 has also
been shown under some circumstances to down-regulate IL-12 production
(29, 30). Neither of these cytokines is readily detected in the serum
after infection. However, when 2 mg of mAb to IL-10 were injected i.p.
4 h before infection with 2 x 104 Listeria, bacterial numbers 5 days later in groups of
5 mice were depressed from 4.86 ± 0.36 in the spleen of untreated
mice to 4.04 ± 0.35 in treated mice
(p < 0.01) and in the liver from 6.18 ± 0.20
to 4.46 ± 0.49 (p < 0.001), showing both that
IL-10 was produced and that our Ab treatment regimen was effective.
Similar treatment with anti-IL-4 mAb caused a depression from the same
baseline numbers to 3.59 ± 0.20 (p < 0.001)
and 5.28 ± 0.48 (p < 0.01) in spleen and
liver, respectively. Presumably, there is more effective macrophage
activation in the absence of these cytokines, leading to the decrease
in bacterial numbers.
To test the effect of mAbs on the course of IL-12 production during
infection, mice were infected with a sublethal dose of
Listeria (2 x 104) with or without
pretreatment with a mixture of 2 mg each of anti-IL-10 and
anti-IL-4 mAbs. Table VI shows that
depleting IL-10 and IL-4 neither enhanced peak production of IL-12 nor
prolonged its production beyond the 24-h peak. At these earlier time
points, the effect on bacterial numbers was less than that observed at
5 days (above) but still significant. We also tested the ability of
these Abs to enhance IL-12 production following injection of
109 heat-killed Listeria, but bioactive IL-12
remained undetectable in serum and spleen homogenates under these
circumstances (results not shown).
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As well as failing to deliver a positive signal for IL-12
production, such as TNF, killed bacteria may deliver a suppressive
signal. Therefore, mice were injected with 109 heat-killed
Listeria intervals before infection with 2 x
104 viable Listeria organisms. Other mice
received live Listeria only. As shown in Table VII, killed bacteria given up to 7 days
before challenge with live Listeria suppressed both IL-12
production measured at 24 h and IFN- production recalled at 10
days postinfection. In other experiments (not shown), the suppressive
effect lasted up to 14 days. Killed bacteria given 28 days before
infection had no effect on the subsequent response to live bacteria.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IL-12
within hours of injection, with TNF mRNA appearing perhaps a little
ahead of the others (12). None of these cytokines was triggered by high
doses of killed Listeria or of Listeria that
could not persist in the host because they lacked the listeriolysin
virulence determinant. It appears from the results of the present
experiments that both positive and negative forces play a role in
controlling the production of IL-12 in response to live or dead
bacteria. These experiments show that, in vivo, live but not killed Listeria and Brucella organisms triggered the secretion of detectable IL-12 bioactive protein. The findings confirm and extend those of others with Listeria (12) and Mycobacterium (33), which used mRNA expression to indicate IL-12 production. However, the in vivo findings conflict with the many observations that IL-12 can be elicited from macrophages in vitro by killed bacteria (13-15) and even by phagocytosis of latex beads (16, 17). Although our experiments suggest that live bacteria are a more efficient stimulus for IL-12 production by macrophages in vitro, the difference was not absolute, in that killed bacteria triggered appreciable IL-12 production. In contrast, the difference between live and killed bacteria in TNF induction in similar experiments was much clearer, with killed bacteria not inducing detectable TNF (20). In addition, the inability of killed mycobacteria to induce TNF production by macrophages in vitro has been reported by Ladel et al. (17). We therefore asked whether IL-12 production was deficient in knockout mice lacking both the type 1 and type 2 TNF receptors.
We found that IL-12 production in response to either
Listeria or Brucella organisms was markedly
deficient in vitro. In vivo, IL-12 was undetectable in serum following
infection of TNFR-/- mice with Listeria, and peak levels
of IL-12 were more than halved compared with wild-type mice following
Brucella infection. Listeria-infected TNFR-/-
mice did not survive long enough to measure the effect on IFN-
production. However, following infection with Brucella, they
showed no deficiency in later IFN- production by T lymphocytes. This
presumably reflects the residual IL-12 in these mice, since we have
previously shown (19) that depletion of IL-12 by injection of mAb
abrogates IL-12 production. The contrast between the total absence of
IL-12 in Listeria-infected TNFR-/- mice vs measurable
IL-12 remaining in the Brucella-infected TNFR-/- mice may
reflect different pathways of stimulation of IL-12 due to LPS in the
Brucella organisms.
Contradictory findings concerning the influence of TNFR deletion on IL-12 production during infection have been published. Endres et al. (34) found that IL-12 mRNA expression by Listeria-infected mice lacking the p55 type 1 TNFR was unaffected. In contrast, Flesch et al. (33), also using type 1 receptor-deficient mice, in this case infected with Mycobacterium bovis BCG, found a marked depression in IL-12 mRNA in vivo and an inability of macrophages in vitro to produce IL-12 p40 protein measured by ELISA. The present findings place us firmly in the latter camp, although it is not easy to explain the difference from the data of Endres at al. Perhaps it relates to our use of bioassay to detect IL-12, rather than the highly sensitive RT-PCR, to detect IL-12 mRNA, or the fact that we used mice lacking both TNF receptors.
TNF may be acting by at least two pathways to promote the early
production of IL-12. First, TNF secreted by the macrophages may act in
an autocrine fashion to up-regulate IL-12 production by macrophages.
TNF together with bacterial products added to macrophages promotes
secretion of IL-12 (33, 35). Secondly, TNF synergizes with IL-12 to
activate NK cells to produce IFN- (36) and IFN- itself
up-regulates IL-12 production by macrophages (33, 37). Live
Listeria organisms are more efficient than killed ones at
activating NK cells in vitro and in vivo (38), perhaps reflecting the
efficiency with which they trigger TNF production.
The relative importance of TNF and IFN- in regulating IL-12
production may be gauged by considering IFN- knockout mice where
IL-12 production is unaffected in a number of systems (39, 40). In
IFN- receptor knockout mice unable to respond to IFN-, there is a
normal IFN- response to infection with Leishmania (41)
and pseudorabies virus (42), implying that in these systems IFN-
does not control its own production, either directly or indirectly
via IL-12.
A notable feature of the IL-12 production during infection was that it was short-lived and ceased while the infecting bacteria were still present in high numbers. It has been said that IL-12 being a strongly inflammatory cytokine must be down-regulated before tissue damage occurs. In a number of systems, this regulation appears to be mediated by IL-10 (11). However, we were unable to enhance or prolong the production of IL-12 by injecting Ab to neutralize IL-10. Nor was Ab to IL-4 able to affect the production of IL-12. This failure of anti-IL-10 and anti-IL-4 applied both to the duration of IL-12 produced in response to live Listeria organisms and to the absence of IL-12 following the injection of heat-killed Listeria organisms. The failure of Ab treatment can always be attributed to inadequate dosage, but the Abs at the doses used were able to affect other functions in the animals, namely numbers of bacteria.
On the other hand, injection of heat-killed Listeria
suppressed the IL-12 response to subsequent exposure to live
Listeria. The suppressive effect lasted longer than 7 days
between injection of heat-killed bacteria and exposure to live
organisms. Not only was the IL-12 response suppressed but also the
production of IFN- by spleen cells taken 7 days after infection and
cultured in vitro with recall Ag. Past experience (43) suggests that
most of the IFN- produced under these conditions comes from
CD4+ T cells (43). Although injection of killed
Listeria only 24 h before live organisms somewhat
suppressed bacterial numbers recovered at the time of IL-12 assay, at
longer intervals between killed bacteria and live, there was no effect
on the numbers of live bacteria recovered.
Although this suppressive effect is reminiscent of the effect of killed bacteria in vitro inducing IL-10 to suppress stimulation of IL-12 production by live bacteria (18), depletion of IL-10, or indeed of IL-4, by injected Ab did not enhance IL-12 production. It is possible that some other suppressive cytokine is involved. The fact that the phenomenon lasts for at least 7 days implies a persistent production of that factor. On the other hand, a mechanism similar to LPS tolerance may apply, where prior exposure to the stimulus renders the macrophages recalcitrant to further stimulation (44).
These data emphasize again the complexity of cytokine interactions
controlling the induction of ACR to protect against intracellular
bacteria. Three observations suggest a key role for TNF in induction of
ACR: TNF is induced by stimulation of resident macrophages by living
but not killed bacteria (17, 20); TNF is a key cytokine in the
activation of NK cells to produce IFN- (38) and, finally, the
present results show the dramatic diminution of bioactive IL-12 during
infection of TNFR-/- mice.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Christina Cheers, Department of Microbiology, University of Melbourne, Parkville, Victoria 3055, Australia. E-mail address:
3 Abbreviations used in this paper: ACR, acquired cellular immunity; HBA, horse blood agar; HKB, heat killed brucellae; HKL, heat killed listeriae.
Received for publication October 30, 1997. Accepted for publication April 7, 1998.
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Y. He, R. Vemulapalli, and G. G. Schurig Recombinant Ochrobactrum anthropi Expressing Brucella abortus Cu,Zn Superoxide Dismutase Protects Mice against B. abortus Infection Only after Switching of Immune Responses to Th1 Type Infect. Immun., May 1, 2002; 70(5): 2535 - 2543. [Abstract] [Full Text] [PDF]
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V. Jubier-Maurin, R.-A. Boigegrain, A. Cloeckaert, A. Gross, M.-T. Alvarez-Martinez, A. Terraza, J. Liautard, S. Kohler, B. Rouot, J. Dornand, et al. Major Outer Membrane Protein Omp25 of Brucella suis Is Involved in Inhibition of Tumor Necrosis Factor Alpha Production during Infection of Human Macrophages Infect. Immun., August 1, 2001; 69(8): 4823 - 4830. [Abstract] [Full Text] [PDF]
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M. S. Rolph and S. H. E. Kaufmann CD40 Signaling Converts a Minimally Immunogenic Antigen into a Potent Vaccine Against the Intracellular Pathogen Listeria monocytogenes J. Immunol., April 15, 2001; 166(8): 5115 - 5121. [Abstract] [Full Text] [PDF]
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V. Nagabhushanam and C. Cheers Non-Major Histocompatibility Complex Control of Antibody Isotype and Th1 versus Th2 Cytokines during Experimental Infection of Mice with Mycobacterium avium Infect. Immun., March 1, 2001; 69(3): 1708 - 1713. [Abstract] [Full Text] [PDF]
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 M. J. Walter, N. Kajiwara, P. Karanja, M. Castro, and M. J. Holtzman Interleukin 12 p40 Production by Barrier Epithelial Cells during Airway Inflammation J. Exp. Med., January 29, 2001; 193(3): 339 - 352. [Abstract] [Full Text] [PDF]
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N. Soussi, G. Milon, J.-H. Colle, E. Mougneau, N. Glaichenhaus, and P. L. Goossens Listeria monocytogenes as a Short-Lived Delivery System for the Induction of Type 1 Cell-Mediated Immunity against the p36/LACK Antigen of Leishmania major Infect. Immun., March 1, 2000; 68(3): 1498 - 1506. [Abstract] [Full Text] [PDF]
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A. Gross, A. Terraza, S. Ouahrani-Bettache, J.-P. Liautard, and J. Dornand In Vitro Brucella suis Infection Prevents the Programmed Cell Death of Human Monocytic Cells Infect. Immun., January& |
