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*
Department of Surgery, Klinikum rechts der Isar, and
Institute of Medical Microbiology, Immunology and Hygiene, Technische Universität, Munich, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-dependent Th1 responses. Here, we
demonstrate that challenging mice with CpG-ODN substantially increases
the resistance against acute polymicrobial sepsis. Systemic levels of
IL-12, IL-18, and IL-10 were not altered in CpG-ODN-treated mice as
compared with controls. In contrast, administration of CpG-ODN resulted
in a strongly enhanced accumulation of neutrophils at the primary site
of infection. Neutrophils of CpG-ODN-treated mice exhibited an
up-regulation of phagocytic receptors, an increased phagocytic
activity, and an elevated production of reactive oxygen metabolites.
These results suggest that the protective effects of CpG-ODNs in
acute polymicrobial sepsis are related to an enhanced effector cell
response of innate immunity. CpG-ODN may therefore represent potent
agents for the treatment of sepsis-associated
immunoparalysis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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was applied to sepsis patients with severely reduced
monocyte expression of MHC class II proteins (11).
Bacterial DNA is discriminated from host-derived self DNA by pattern
recognition systems of the innate immune system (12, 13, 14).
Unmethylated CpG dinucleotides in the context of particular flanking
sequences (CpG motifs) are regarded as the structural basis for this
specific recognition. In contrast to prokaryotic DNA, vertebrate DNA
exhibits a low abundance of CpG dinucleotides and a high degree of
cytosine methylation. Bacterial DNA and oligodeoxynucleotides
(ODN)3 containing CpG
motifs (CpG-ODN) trigger macrophages and dendritic cells to up-regulate
MHC class II and costimulatory molecules and to secrete inflammatory
cytokines such as TNF, IL-1, IL-6, and IL-12 (15, 16, 17, 18, 19, 20). CpG
DNA also activates humoral immunity by inducing B cell proliferation
and secretion of IL-6 and IgM (21, 22). Cell activation by
CpG DNA requires the endosomal uptake of DNA and the rapid stimulation
of the stress kinase pathways and NF-
B (16, 23).
Moreover, CpG DNA augments the IL-12-stimulated production of IFN-
by NK cells (24). Recent reports also show that CpG DNA
acts as strong adjuvant to promote Ag-specific responses and to enhance
protective Th1 immune reactions in models of chronic infection with
intracellular bacteria and parasites as well as viruses
(25, 26, 27, 28, 29, 30). However, excessive or inappropriate exposure to
CpG DNA may also result in adverse effects such as septic shock and
pulmonary inflammation (15, 16, 31).
The present study investigated the effects of CpG-ODN administration on the immune response against acute polymicrobial sepsis. We demonstrate that pretreatment of mice with CpG-ODN substantially improves host defense and that the protective effects of CpG-ODNs in this model are associated with an enhanced effector cell response of innate immunity. Thus, DNA sequences containing CpG motifs may represent valuable and potent agents for the treatment of sepsis-associated immunosuppression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Phosphothioate modified ODN were custom synthesized by MWG Biotec (Munich, Germany). The sequence of the CpG-ODN equals ODN 1668 and was 5'-TTCATGACGTTCCTGATGCT-3' (21). The sequence of the control ODN (5'-GCTTGATGACTCAGCCGGAA-3') does not contain CpG motifs and equals ODN AP-1 (32). The control ODN was chosen, because it did not show any biological activity in various experimental systems tested (32, 33). Specific Abs and isotype-matched Ig controls were purchased from PharMingen (Hamburg, Germany) or Serotec (Darmstadt, Germany). In this study, rat Abs against murine Ly6G/Gr-1 (RB6-8C5), Mac-1 (M1/70), CD16/CD32 (2.4G2), and CD13 (R3-242) were used. LPS from Escherichia coli (serotype O127:B8) and PMA were obtained from Sigma (St. Louis, MO).
Animal model of acute polymicrobial peritonitis
Female BALB/c mice were purchased from Harlan Winkelmann GmbH
(Borchen, Germany). Mice were bred and maintained under specific
pathogen-free conditions. Experimental animals were used at 8–10 wk of
age. To induce polymicrobial sepsis, the colon ascendens stent
peritonitis (CASP) procedure was performed as described previously
(34). Briefly, a 16-gauge venous catheter was prepared by
creating a notch at a distance of 2 mm from the orifice. One millimeter
beyond, the catheter was cut with a scalpel. Following laparotomy, the
colon ascendens was exteriorized and a 7/0 ethilon thread (Ethicon,
Nordersted, Germany) was stitched through the antimesenteric portion of
the colon ascendens 
10 mm distal of the ileocecal valve. The
prepared venous catheter was punctured antimesenterically through the
colonic wall, proximal of a pretied knot, and fixed. A second dication
was performed proximal of the stent. The inner needle was removed and
the stent was cut at the prepared site. To ensure proper intraluminal
position of the stent, stool was milked from the cecum into the colon
ascendens, until a drop of about 1 mm in diameter appeared. Fluid
resuscitation of animals was performed by flushing 0.5 ml of sterile
saline solution into the peritoneal cavity before closure of the
abdominal wall.
The CASP procedure was developed as a model for postoperative septic
peritonitis. It was shown that survival of CASP is dependent on IFN-
and IL-12 (9, 34). Importantly, investigation of surgical
patients revealed a direct correlation between the development and
mortality of postoperative intraabdominal infection and the monocyte
production of IL-12 (7, 9). These observations therefore
suggest that the CASP model reflects important aspects of host defense
during postoperative intraabdominal infection.
Treatment protocol
ODNs were dissolved in PBS and 10 nmol (64 µg) were injected i.p. 6 days before ex vivo analysis or CASP surgery. In some control experiments, mice received PBS 6 days before analysis.
Previous work has shown that the protective effects of CpG-ODN pretreatment develop within 2–3 days and persist for at least 2 wk (25, 26, 27, 28). Therefore, we argued that alterations of the resistance against septic peritonitis may have developed by day 6 and would persist throughout the experiments.
Bacterial load of peripheral organs
Mice were sacrificed 20 h after CASP surgery, and peritoneal lavage fluid was collected. Lungs, livers, and spleens were removed and homogenized in 10 ml sterile PBS. Serial dilutions of organ homogenates in PBS were plated on blood agar plates (Becton Dickinson, Heidelberg, Germany). CFU were counted after incubation at 37°C for 24 h and calculated as CFU per whole organ.
Cytokine analysis
Serum samples were collected either 6 days after ODN treatment or 20 h after CASP surgery. Spleen cell suspensions were plated on plastic dishes, and adherent cells were collected after incubation at 37°C for 1 h. Adherent spleen cells (1 x 106) were stimulated either with 50 ng/ml LPS, 1 µM (6.4 µg/ml) CpG-ODN, or 1 µM (6.4 µg/ml) control ODN for 16 h, and supernatants were collected. Cytokine levels in serum or supernatants were determined by specific ELISA according to the manufacturer’s protocols. Except for IL-10 (Endogen, Woburn, MA), all ELISA kits were purchased from R&D Systems (Minneapolis, MN). The levels of sensitivity were 5 pg/ml for TNF, 4 pg/ml for IL-12, 8 pg/ml for IL-18, and 12 pg/ml for IL-10.
Flow cytometry
For analysis of peripheral blood leukocytes, heparinized blood was collected, erythrocytes were lysed for 5 min in lysis buffer (155 mM ammonium chloride, 15 mM sodium bicarbonate, 1 mM EDTA), and cells were used for Ab staining. In addition, peritoneal exudate cells were analyzed. Cells were incubated with fluorochrome-conjugated mAbs for 30 min at 4°C in PBS containing 1% BSA. In each experiment, appropriate isotype-matched controls were included. After washing with PBS fluorescence of unfixed cells was analyzed on an EPICS XL flow cytometer (Coulter, Hialeah, FL).
Oxidative burst and phagocytosis
Production of oxygen metabolites was assessed by a flow cytometric method as described (35). Briefly, heparinized blood from CpG and control ODN-treated mice was collected, and erythrocytes were lysed as described above. Leukocytes were washed in HBSS and loaded for 15 min at 37°C with 60 µM dihydrorhodamine 123. After addition of 2.5 µM sodium azide and 0.05 mM cytochalasin B, the cells were incubated with 500 ng/ml PMA for 20 min at 37°C and flow cytometry analysis was performed immediately thereafter. Alternatively, leukocytes were incubated with FITC-labeled and opsonized E. coli (Orpegen Pharma, Heidelberg, Germany) at 37°C for 10 min. Thereafter, fluorescence of bacteria adhering to the cells was quenched, and cells were washed and fixed with 0.1% paraformaldehyde. Phagocytosis rate was measured by flow cytometry using a Epics XL cytometer (Coulter, Hialeah, FL).
Statistical analysis
Statistical analysis of the data was performed using the Student’s t test except for survival data, which were analyzed using the log rank test. The level of significance was set at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether treatment of mice with CpG-ODN modulates the
host defense against acute polymicrobial peritonitis, mice were
injected with 10 nmol CpG-ODN or control ODN, and, 6 days later, the
CASP procedure (34) was performed. The results in Fig. 1
show that survival of CpG-ODN-treated
mice was significantly improved compared with mice treated with control
ODN (n = 18 for CpG-ODN treatment and n
= 17 for controls). Survival of control ODN-treated mice, however, was
not significantly different from mice receiving PBS (n
= 12) as determined by log rank test (p =
0.626). To further corroborate the protective effects of CpG-ODN, the
bacterial clearance in the peritoneal cavity and in peripheral organs
(spleen, lungs, and liver) of CpG and control ODN-treated mice was
determined. We observed that, 20 h after CASP, significantly lower
numbers of viable bacteria were present in peritoneal cavity, spleen,
and lungs of mice injected with CpG-ODN as compared with controls (Fig. 2). Although the bacterial numbers in the
livers of CpG-ODN-treated mice were about 2-fold lower that in control
mice, this difference did not reach statistical significance (Fig. 2).
Collectively, these results demonstrate that treatment of mice with
CpG-ODN induces a state of increased resistance to acute polymicrobial
peritonitis.
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CpG-ODN exhibit protective effects in models of chronic infection
with intracellular pathogens that are related to increased Th1
responses (25, 26, 27, 28, 29, 30). In addition, survival of acute
polymicrobial sepsis in the CASP model was shown to be dependent on
IFN- (34). Therefore, we analyzed the effects of
CpG-ODN treatment on the systemic levels of the IFN--regulating
cytokines IL-12 and IL-18 as well as on IL-10 production 20 h
after CASP. As depicted in Table I,
baseline serum levels of IL-12, IL-18, and IL-10 were not significantly
different between CpG and control ODN-treated mice. CASP surgery
up-regulates production of IL-10 and IL-18, but not of IL-12 in both
treatment groups (Table I). Again, no significant difference was
observed between CpG-ODN-treated mice and controls (Table I). Serum
levels of IFN- were below the limits of detection in both groups
(data not shown). These results show that CpG-ODN treatment does not
lead to alterations of systemic cytokine levels in response to acute
polymicrobial sepsis.
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To further elucidate potential mechanisms of immune protection by
CpG-ODN treatment, the local inflammatory response was examined.
CpG-ODN administration without additional infectious stimulus caused a
moderate increase in total leukocyte numbers or
Gr-1+ cells (Fig. 3). Leukocyte counts in peripheral blood
were not altered by CpG-ODN or control ODN treatment (data not shown).
When the CASP procedure was performed, peritoneal leukocyte
accumulation was observed in both treatment groups 20 h later. It
should be noted, however, that cell numbers were strongly elevated in
CpG-ODN-treated mice as compared with controls (Fig. 3A).
Fig. 3B shows that in both groups most of the accumulating
cells were neutrophils as identified by the expression of both Gr-1 and
Mac-1 (36). Thus, 20 h after CASP, there were about
4-fold more neutrophils in the peritoneal cavities of CpG-ODN than of
control ODN-treated mice.
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, administration of
CpG-ODN also affected the expression of phagocyte receptors on
peritoneal neutrophils. Expression of CD16/CD32, Mac-1, and CD13 was
elevated on Gr-1+ peritoneal cells of
CpG-ODN-treated mice when compared with controls. Expression of
CD16/CD32 and Mac-1, but not of CD13 was further increased 20 h
after CASP surgery (Fig. 4, A and B).
Importantly, the increase in CD16/CD32 and Mac-1 expression following
CASP was significantly greater in CpG-ODN than in control ODN-treated
mice (Fig. 4, A and B). Fig. 4C
depicts representative histograms for Mac-1 expression on peritoneal
Gr-1+ cells following CASP, showing increased
surface density of Mac-1 in CpG-ODN-treated mice as compared with
controls. These results indicate that treatment of mice with CpG-ODN
leads to an up-regulation of neutrophil phagocytic receptors at the
primary site of infection.
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To determine the effects of CpG-ODN treatment on the antimicrobial
functions of neutrophils, mice were injected with CpG-ODN or control
ODN and, 6 days later, peripheral blood neutrophils were examined for
their phagocytic capacity and their production of reactive oxygen
metabolites. In these experiments, neutrophils were identified by their
forward and side scatter profiles (Fig. 5A) to avoid a possible
interference of Gr-1 and Mac-1 Abs with the functional assays. The
results in Fig. 5B demonstrate that peripheral blood
neutrophils of CpG-ODN-treated mice exhibited a significantly increased
phagocytic capacity for opsonized E. coli when compared with
cells from control mice. Moreover, the PMA-stimulated production of
reactive oxygen metabolites by neutrophils was significantly greater
following CpG-ODN than control ODN treatment of mice (Fig. 5C). Thus, priming of mice with CpG-ODN results in an
improved antimicrobial activity of circulating neutrophils. Together
with the results depicted in Figs. 3
and 4, these findings indicate
that CpG-ODN treatment of mice leads to an amplification of the innate
effector cell response against acute polymicrobial sepsis.
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Pretreatment with LPS renders mice resistant against the toxic
effects of a challenge with high doses of LPS (37). In
addition, endotoxin tolerant mice are partially protected from the
lethality induced by septic peritonitis (38, 39). Because
the biological responses triggered by CpG-ODN are similar to those
induced by LPS, we investigated whether treatment with CpG-ODN would
alter cytokine production by mononuclear phagocytes. Mice were injected
i.p. with PBS, LPS, CpG-ODN, or control ODN, and, 4–6 days later,
spleens were removed. The plastic adherent fraction of splenocytes was
restimulated in vitro with LPS, CpG-ODN, or control ODN, and the
secretion of TNF and IL-12 was determined. The results in Fig. 6A indicate that TNF
production of cells from mice challenged with endotoxin is diminished
both after LPS and CpG-ODN restimulation. In contrast,
adherence-purified spleen cells from CpG-ODN-treated mice showed an
increased TNF release after CpG-ODN stimulation (Fig. 6A).
Similar results were obtained for IL-12 secretion (Fig. 6B).
These results show that endotoxin desensitizes adherent splenocytes
against CpG-ODN-induced signals. However, treatment of mice with
CpG-ODN does not induce tolerance against CpG-ODN or cross-tolerance
against LPS stimulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by cells of the innate immune system, which in turn
may promote protective Th1 immune reactions in response to infection.
The present report extends these studies and demonstrates that
challenge of mice with CpG-ODN induces immune protection against acute
polymicrobial sepsis. The protective effects were found to be
associated with an augmented accumulation of neutrophils at the primary
site of infection and an enhanced antimicrobial activity of these
cells. The systemic levels of IL-12 and IL-18, however, were not
affected by CpG-ODN administration. Although locally restricted
alterations of IFN- production cannot be excluded, these results
suggest that the protective effects of CpG-ODN are not mediated by an
enhanced Th1 response. Moreover, the rapid progression of infection and
early mortality in the CASP model (34, 35) also indicate
that host defense is mainly dependent on effector mechanisms of the
innate rather than the adaptive immune system. Collectively, our
observations provide strong evidence that the immune modulating
activities of CpG-ODN are not restricted to the instruction and
reprogramming of adaptive immunity but, in addition, may involve the
amplification of the innate effector cell response during acute
polymicrobial infection.
Polymicrobial sepsis induced by CASP leads to a rapid recruitment of
neutrophils into the peritoneal cavity (34). CpG-ODN
treatment was shown to augment this neutrophil accumulation by about
4-fold. In addition, administration of CpG-ODN increased the surface
expression of Mac-1 and Fc receptors on resident peritoneal
neutrophils and strongly enhanced the up-regulation of these receptors
following CASP. Therefore, it is conceivable that the increase in
neutrophil numbers and the elevated surface density of phagocytic
receptors contribute to the improved bacterial clearance, which was
observed in mice challenged with CpG-ODN. Thus, up-regulation of Mac-1
and Fc-receptors may improve phagocytosis of opsonized bacteria by
neutrophils in CpG-ODN-treated mice and increased cell surface Mac-1
may facilitate cell recruitment from the circulation to the inflamed
peritoneal cavity (40, 41).
In addition to phagocytosis of microorganisms, the production of antimicrobial substances, such as reactive oxygen metabolites, is a prerequisite for clearing bacterial infections (42). The importance of reactive oxygen intermediates for host defense is clearly demonstrated in patients suffering from chronic granulomatous disease, which is characterized by severe and life-threatening infections with bacteria and fungi (43, 44). Chronic granulomatous disease is caused by diverse mutations of components of the NAPDH oxidase, thereby abolishing the oxidative burst reaction of phagocytes (45). The mechanisms of immune pathology that underlie this genetic disorder were confirmed in mice deficient for p47phox (46). It was therefore interesting to note that circulating neutrophils of mice treated with CpG-ODN showed an enhanced production of reactive oxygen metabolites after stimulation with PMA. Considered together, these studies support the view that an increased oxidative burst activity of neutrophils contributes to the protective effects of CpG-ODN in acute polymicrobial sepsis.
Challenging mice with CpG-ODN results in extramedullary hematopoesis and transient splenomegaly peaking at day 6 after injection (33). Spleen enlargement was associated with increased frequencies of B220/CD3 double-negative cells that contained myeloid and erythroid progenitor cells (33). In addition, CpG-ODN administration was found to accelerate the recovery of cytotoxic T cell responses and the resistance against Listeria monocytogenes infection after sublethal irradiation (33). In the present report, we show that treatment of mice with CpG-ODN alters the phenotype and function of neutrophils with an up-regulation of phagocytic receptors and an increased capacity to phagocytose bacteria and to produce reactive oxygen metabolites. Thus, the hematopoietic activities of CpG-ODNs might also cause alterations of the differentiation or activation state of neutrophils leading to these phenotypic changes. It is therefore conceivable that CpG-ODN may enhance immune defense against polymicrobial sepsis as a consequence of their effects on hematopoesis.
The biological responses triggered by CpG-ODNs are similar to those induced by LPS. For example, macrophage activation by CpG-ODNs and LPS involves similar signaling pathways (23, 47, 48) and results in the production of multiple inflammatory cytokines, which may cause septic shock in sensitized mice (15, 16). Because treatment of mice with LPS tolerizes against the pathologic effects of a subsequent challenge with high dose LPS (37) and protects from lethality of septic peritonitis (38, 39), we asked whether treatment of mice with CpG-ODN would lead to a similar state of reduced macrophage responsiveness. The results of the present report demonstrate, however, that challenging mice with CpG-ODN does not induce tolerance to a subsequent stimulation with CpG-ODN nor does it generate cross-tolerance with LPS. In contrast, LPS was shown to tolerize macrophages against CpG-ODN-mediated signals. These findings therefore suggest that despite certain similarities the in vivo response of macrophages to LPS and CpG-ODN also exhibits substantial differences and that the protective effects of CpG-ODN are not related to the induction of a functional status resembling LPS tolerance.
Previous work has shown that bacterial DNA and ODN containing CpG motifs may cause a TNF-dependent septic shock in mice that were sensitized with D-galactosamine (15, 16). These observations are reminiscent of studies demonstrating that lethal TNF-mediated shock is triggered by the injection of bacterial toxins such as LPS or staphylococcal enterotoxin B into sensitized mice (49, 50). However, several lines of evidence suggest that these toxin models do not correctly reflect the major pathogenic events in human sepsis. Several clinical trials failed to demonstrate beneficial effects of antiinflammatory treatment strategies or reported even adverse effects of mediator neutralization (1, 2, 3, 4, 5). Patient investigations have further indicated that an impaired cytokine production of monocytes and T cells is associated with an increased incidence or mortality of sepsis (6, 7, 8, 9, 10, 51). Moreover, mouse models of peritoneal sepsis have directly demonstrated essential protective functions of endogenously produced or exogenously administered proinflammatory cytokines (10, 34, 52, 53, 54, 55, 56). Collectively, these observations support the notion that immunosuppression may represent the major pathogenic process in sepsis and that therefore beneficial effects may be obtained by stimulating the proinflammatory response. The results of the present report indicating that pretreatment of mice with CpG-ODN enhances the neutrophil response and improves outcome of septic peritonitis are consistent with this concept. Nevertheless, it should be noted that an increased inflammatory response has the potential to result in adverse effects such as organ injury and shock. It is therefore conceivable that outcome is affected by a critical balance between antimicrobial and autoaggressive effects of CpG-ODN treatment. Although this balance may vary depending on the type of infection or the type of immune response triggered, our data argue that the protective effects predominate in sepsis.
In summary, the present report demonstrates that administration of CpG-ODN increases the resistance against acute polymicrobial sepsis and that immune protection is associated with an enhanced effector cell response of innate immunity. Thus, our study suggests that CpG-ODNs may represent inexpensive and potent agents for the treatment of sepsis-associated immunoparalysis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bernhard Holzmann, Department of Surgery, Klinikum rechts der Isar, Technische Universität, Ismaninger Strasse 22, 81675 München, Germany.
3 Abbreviations used in this paper: ODN, oligodeoxynucleotide; CASP, colon ascendens stent peritonitis.
Received for publication May 4, 2000. Accepted for publication July 24, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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interferon in survival of colon ascendens stent peritonitis, a novel murine model of abdominal sepsis. Infect. Immun. 66:2300.This article has been cited by other articles:
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M. P. Gould, J. A. Greene, V. Bhoj, J. L. DeVecchio, and F. P. Heinzel Distinct Modulatory Effects of LPS and CpG on IL-18-Dependent IFN-{gamma} Synthesis J. Immunol., February 1, 2004; 172(3): 1754 - 1762. [Abstract] [Full Text] [PDF]
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C. I. Ojielo, K. Cooke, P. Mancuso, T. J. Standiford, K. M. Olkiewicz, S. Clouthier, L. Corrion, M. N. Ballinger, G. B. Toews, R. Paine III, et al. Defective Phagocytosis and Clearance of Pseudomonas aeruginosa in the Lung Following Bone Marrow Transplantation J. Immunol., October 15, 2003; 171(8): 4416 - 4424. [Abstract] [Full Text] [PDF]
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A. Iwata, V. M. Stevenson, A. Minard, M. Tasch, J. Tupper, E. Lagasse, I. Weissman, J. M. Harlan, and R. K. Winn Over-Expression of Bcl-2 Provides Protection in Septic Mice by a trans Effect J. Immunol., September 15, 2003; 171(6): 3136 - 3141. [Abstract] [Full Text] [PDF]
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Y. Wang and A. M. Krieg Synergy between CpG- or non-CpG DNA and specific antigen for B cell activation Int. Immunol., February 1, 2003; 15(2): 223 - 231. [Abstract] [Full Text] [PDF]
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J. H. Wang, M. Doyle, B. J. Manning, S. Blankson, Q. D. Wu, C. Power, R. Cahill, and H. P. Redmond Cutting Edge: Bacterial Lipoprotein Induces Endotoxin-Independent Tolerance to Septic Shock J. Immunol., January 1, 2003; 170(1): 14 - 18. [Abstract] [Full Text] [PDF]
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B. K. Choudhury, J. S. Wild, R. Alam, D. M. Klinman, I. Boldogh, N. Dharajiya, W. J. Mileski, and S. Sur In Vivo Role of p38 Mitogen-Activated Protein Kinase in Mediating the Anti-inflammatory Effects of CpG Oligodeoxynucleotide in Murine Asthma J. Immunol., November 15, 2002; 169(10): 5955 - 5961. [Abstract] [Full Text] [PDF]
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, n = 18) or control ODN (
,
n = 17) 6 days before CASP. Survival was monitored
over a period of 14 days. Three independent experiments were performed,
and the data were pooled. Each experiment demonstrated an improved
survival of CpG-ODN-treated mice as compared with controls. Statistical
significance was determined using the log rank test.
) or control ODN (
), or stimulated in vitro with
control ODN (
) for 16 h.
Production of TNF (A) or IL-12 (B) was
quantitated by ELISA. Results are presented as mean ± SEM and are
derived from four independent experiments each using cells pooled from
three individual mice per group. *, p < 0.01 and
#, p < 0.05 (vs medium control and control ODN of
the same treatment group).