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Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-cells of the pancreas (1). Several
Ags have been identified as targets for diabetogenic T cells, including
-cell-specific proteins such as insulin, non--cell-restricted Ags
such as glutamic acid decarboxylase (GAD), and even ubiquitous Ags such
as 60-kDa heat shock protein (HSP60) (1). It has been
shown that the onset of diabetes is preceded by an increase in T cell
reactivity toward HSP60 and to an HSP60 peptide contained between aa
437 and 460 named p277 (2). In contrast to the early T
cell reactivity, Abs to HSP60 and p277 can only be detected late in the
natural history of the disease, months after the onset of clinical
diabetes when the destructive process has terminated (3).
Peptide p277 administered to NOD mice in IFA can arrest the development
of diabetes (4). Furthermore, p277 treatment is able to
induce remission of advanced insulitis even after the clinical onset of
hyperglycemia (5). Successful treatment is associated with
down-regulation of spontaneous T cell reactivity to p277 and the
induction of Abs to p277; these Abs have Th2-associated isotypes (IgG1
and IgG2b), otherwise not found in young NOD mice (6, 7). However, peptide therapy is only one way to modulate an ongoing autoimmune process. DNA vaccination is also an efficient approach to induce protection against infectious pathogens (8) and cancer (9) and to modulate autoimmune processes (10). It has been shown that after i.m. injection of a naked expression vector, plasmid DNA is taken up by muscle cells and maintained episomally, allowing the expression of the encoded Ag (11). Thus, after single or repeated injections of DNA, cellular and/or humoral immune responses to the encoded protein are mounted, and long-lived memory lymphocytes are induced (12). These memory cells may have regulatory functions and, therefore, might serve as tools for the modulation of autoimmune conditions.
To explore the potential of a DNA-based therapy of diabetes, we set out to investigate whether immunization with a DNA construct encoding for the HSP60 protein could modulate autoimmunity and prevent the onset of the disease. Surprisingly, not only the HSP60-containing construct but also the empty vector (pcDNA3) were capable of reducing the incidence of diabetes. Indeed, the CpG oligonucleotide motif present in the construct could by itself be used to inhibit the development of NOD diabetes. Despite the absence of HSP60, effective treatment was associated with specific immune effects on HSP60 autoreactivity: down-regulation of the spontaneous T cell proliferation to HSP60 and p277 and the induction of specific Abs to these molecules.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female mice of the NOD/LtJ strain were raised and maintained under pathogen-free conditions in the Animal Breeding Center of this institute from breeders supplied by Dr. E. Leiter (The Jackson Laboratory, Bar Harbor, ME). These mice manifest insulitis beginning at about 1 mo of age, which progresses to overt hyperglycemia beginning at about 3 mo of age. The cumulative incidence of IDDM rises to 85% or greater by 6 mo of age. Female BALB/c mice were also raised in this Institute.
Construction of DNA vaccine
The full-length cDNA of human the hsp60 gene was cloned into the pcDNA3 vector (Invitrogen, Leek, The Netherlands) under the control of the human CMV promotor. In brief, hsp60 cDNA in pGEM was amplified using specific oligonucleotides containing restriction sites for the enzyme BamHI or HindIII. The amplicon and the pcDNA3 vector were purified and digested with BamHI/HindIII. The digested PCR product coding for HSP60 and the linearized pcDNA3 vector were ligated using T4 DNA ligase according to the standard protocol provided by the manufacturer. The ligated plasmid was transformed into Escherichia coli and later sequenced to confirm correct insertion of the cDNA (data not shown).
Plasmid preparation and injection
Large-scale plasmid DNA preparations were produced by the
alkaline lysis method using Qiagen Plasmid Mega Prep (Qiagen, Santa
Clarina, CA). DNA was ethanol-precipitated and resuspended in sterile
PBS. Spectrophotometric analysis revealed 260/280 nm ratios 
1.80. The
purity of DNA preparations was confirmed on a 1% agarose gel.
Endotoxin levels were checked by Limulus amebocyte lysate
and were always found to be under acceptable levels for in vivo use
(<0.02 EU/µg DNA).
Eight-week-old NOD or BALB/c females were injected with 100 µl of 10 mM cardiotoxin (Sigma, Rehovot, Israel) into the tibialis anterior muscle using a sterile 27-gauge syringe, witted with a plastic collar to limit needle penetration to 2 mm. Five, 12, and 19 days later the mice were injected with 100 µl (1 µg/µl) of the desired DNA vaccine or with PBS as controls.
Phosphorothioaete oligonucleotides were synthesized at the Oligonucleotide Synthesis Unit of the Weizmann Institute of Science (Rehovot, Israel). One hundred microliters (1 µg/µl) of each preparation was injected as described above following the same time schedule. The oligonucleotide CpG contains two 9-mer segments, which are present in the pcDNA3 ampicillin resistance gene. The control oligonucleotide GpC displays the same nucleotides with an inverted motif: oligonucleotide CpG, 5'-TCCATAACGTTGCAAACGTTCTG-3'; and oligonucleotide GpC, 5'-TCCATAAGCTTGCAAAGCTTCTG-3'.
Blood glucose
Hyperglycemia was defined as a blood glucose level exceeding 13 mM, tested using a Beckman Glucose Analyzer II (Beckman Instruments, Brea, CA).
Peptides and Ags
Peptides were synthesized by a standard F-moc procedure as previously described (5). The peptides were purified by reverse phase HPLC, and their compositions were confirmed by amino acid analysis. The p277 peptide used in this study was VLGGGVALLRVIPALDSLTPANED (2). Insulin and GAD were purchased from Sigma (Rehovot, Israel). Recombinant HSP60 was prepared as described previously (2). Con A was purchased from Sigma.
T cell proliferation
Groups of 8-wk-old female NOD mice received three weekly injections of PBS, pcDNA3, or pHSP60 as described. Four weeks after the last dose the spleens were removed, and the T cell proliferative responses were assayed in vitro in response to the T cell mitogen Con A, the p277 peptide, or HSP60 (13). Dose-response curves were made to establish optimal doses (not shown). The concentration of 10 µg/ml was chosen for the HSP60 protein, 1 µg/ml was chosen for p277, and 1.25 µg/ml was chosen for Con A to illustrate the results, because these concentrations produced the optimum response. T cell responses were detected by the incorporation of [methyl-3H]thymidine added to the wells in quadruplicate cultures for the last 18 h of a 72-h culture. The stimulation index was computed as the ratio of the mean counts per minute of Ag- or mitogen-containing wells to that of control wells cultured without either. The SD from the mean counts per minute was always <10%. Background counts per minute in the absence of Ags ranged from 800-1500.
Cytokine assays
Spleen cells were prepared from 10-wk-old NOD females. The spleen cells were incubated in triplicate with medium alone or with increasing concentrations of CpG or GpC oligonucleotide. Supernatants were collected at 48 h. Cytokines in supernatants were detected by ELISA using PharMingen paired Abs (PharMingen, San Diego, CA), according to the PharMingen cytokine ELISA protocol. PharMingen recombinant mouse cytokines were used as standards for calibration curves. The concentrations of cytokines are shown as the mean nanograms per milliliter derived from calibration curves using recombinant cytokines as standards.
ELISA
Mouse sera were tested for Abs binding to the p277 peptide or HSP60 as previously described (6). Briefly, 10 µg/ml of the various Ags were applied to assay microplates (Maxisorp, Nunc, Roskilde, Denmark), and the plates were incubated with the test sera. The binding of Abs was detected using alkaline phosphatase-conjugated anti-mouse IgG or isotype-specific anti-mouse IgG1, IgG2a, or IgG2b (Jackson ImmunoResearch, West Grove, PA). A significant amount of Ab was defined as an OD405 nm reading >0.25, which is 3 SD above the mean ELISA reading obtained using sera from 10 normal BALB/c mice.
Pancreas histology
Mice from each treatment group were killed at 6 mo of age, when almost all the nontreated mice or control NOD mice were sick. The pancreata were fixed in 10% buffered formalin, cut, and stained by standard hematoxylin and eosin, and the average degree of insulitis was assessed over 20 islets scored per pancreas. The islets where classified as clear when no infiltrate was detected, as mildly infiltrated when peri-insulitis or an intraislet infiltrate occupying <25% of the islet was detected, infiltrated when 25–50% of the islet was occupied by intraislet inflammatory cells, and heavily infiltrated when >50% of the islet was occupied.
Statistical significance
The InStat 2.01 program (GraphPad Software, San Diego, CA) was
used for statistical analysis. Student’s t test and the
2 test were conducted to assay significant
differences between experimental and control groups.
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Results |
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To test whether the pcDNA3 plasmid containing human HSP60, here
named pHSP60, was specifically immunogenic, female BALB/c mice were
injected twice (days 5 and 23) i.m. with 100 µg of pcDNA3 or pHSP60
and assayed periodically for serum Abs. Fig. 1
A shows that the BALB/c mice
immunized with pHSP60 developed specific anti-HSP60 IgG Abs,
whereas no Abs to the HSP60 protein could be detected in those animals
immunized with pcDNA3. Anti-HSP60-specific Abs were detected as early
as 14 days after a single DNA injection (p <
0.02 vs pcDNA3-vaccinated controls). A booster effect was evident 10
days after the second DNA injection (p < 0.05
vs the same group after the first dose; p < 0.005 vs
pcDNA-vaccinated mice). The immune response induced by DNA vaccination
with pHSP60 was specific; pHSP60 did not induce Abs to the nonrelated
recombinant protein GST, as shown in Fig. 1B. These results
demonstrate that the pHSP60 construct, but not the empty pcDNA3 vector,
can induce in BALB/c mice significant amounts of specific Abs after one
vaccination and increasing titers after boosting.
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To test whether immunization with pHSP60 might modulate the
development of spontaneous diabetes in NOD mice, we vaccinated groups
of 8-wk-old female NOD mice, three times at weekly intervals, and
followed their glucose levels. Fig. 2
shows the cumulative incidence of diabetes. Both untreated animals and
those treated with PBS developed the expected incidence of diabetes for
NOD females; 
90% of them were sick by the age of 6 mo. Those
vaccinated with pHSP60 and, to our surprise, also those vaccinated with
the empty pcDNA3 construct showed a significant reduction in the
incidence of diabetes. Only about 41% of those treated with pHSP60
(p < 0.002) and 38% of those treated with
pcDNA3 (p < 0.001) were diabetic at the age of
6 mo. Thus, DNA vaccination modulates the onset of diabetes by a
mechanism that is not associated with the presence of the
hsp60 gene in the administered vector.
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shows that 40–50% of the islets
obtained from the nontreated or PBS-treated mice were heavily
infiltrated, and only 5–10% of the islets were free from insulitis.
In contrast, 50–70% of the islets obtained from DNA-treated mice were
free from insulitis (p < 0.001 for both
pcDNA3-injected mice and the pHSP60 group vs nontreated mice or those
treated with PBS). The differences between the groups treated with
pHSP60 and pcDNA3 were not significant.
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Inhibition of T cell proliferation in response to HSP60 and p277 in DNA-vaccinated mice
The process leading to the onset of diabetes in NOD mice can be
arrested by administration of peptide p277, derived from HSP60
(2). Successful treatment of NOD mice with peptide p277 is
associated with the induction of specific Abs to p277 along with a
decrease in the proliferation of T cells in response to HSP60 and p277
(6). We therefore assayed the splenocytes isolated from
DNA-vaccinated or PBS-treated NOD mice to check their proliferative
responses to p277 and HSP60. As shown in Fig. 4, PBS-treated NOD mice manifested
spontaneous reactivities to HSP60 and p277. In contrast, splenocytes
from the mice vaccinated with pcDNA3 or pHSP60 showed diminished
reactivities to p277 (p < 0.05) and HSP60
(p < 0.01). However, T cells from both treated
and nontreated mice showed similar reactivities to Con A (not shown),
thus indicating that there was no general inhibition of T cell
reactivity induced by DNA vaccination. These results suggested that
treatment with plasmid DNA down-regulated the spontaneous proliferative
response directed to HSP60 and p277 characteristic of the diabetogenic
process in NOD mice.
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The decrease in T cell proliferation in response to HSP60 and its
peptide p277 observed in NOD mice protected from diabetes by treatment
with p277 is associated with the induction of Abs directed to p277
(6). To determine whether the protective effect of DNA
vaccination might be associated with the appearance of Abs to HSP60 and
p277, we analyzed Ab responses in DNA-vaccinated animals 14 days after
the last DNA injection, at the age of 14 wk. Fig. 5A shows that Abs to p277 were
not detected in the sera of untreated or PBS-injected animals. The
absence of Abs to p277 and HSP60 is expected in NOD mice of this age
(3). We also did not detect Abs to p277 in BALB/c mice
immunized with pHSP60, where the appearance of anti-HSP60 Abs was
demonstrated (Fig. 7 and data not shown). However, NOD mice vaccinated
with pHSP60 or pcDNA3 manifested significant levels of Abs to p277
(p < 0.001). Thus, inhibition of diabetes in
NOD mice by DNA vaccination with either pcDNA3 or pHSP60 is associated
with the induction of Abs to HSP60 and peptide p277, even though the
pcDNA3 construct does not contain genetic material encoding HSP60.
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Bacterial DNA contains immunostimulatory sequences that are recognized by the immune system as danger signals and trigger a series of responses in cells of both the innate and adaptive immune system (14, 15, 16). The pcDNA3 vector contains the immunostimulatory CpG sequence in its ampicillin resistance gene (17). We therefore tested whether a DNA oligonucleotide with two CpG sequences could induce the production of specific Abs to HSP60 and p277 that followed vaccination with pcDNA3. As a control we used the oligonucleotide GpC, in which the CpG motifs were inverted.
Eight-week-old NOD mice were treated with oligonucleotide CpG or GpC,
and Abs to HSP60, p277, GAD, insulin, and GST were assayed by ELISA at
the age of 14 wk. As shown in Fig. 5
A, treatment with the
CpG oligonucleotide induced significant levels of Abs to HSP60 and p277
(p < 0.002). Moreover, the titer of Abs
induced by CpG was also significant compared with the levels found in
GpC-treated mice (p < 0.02). Because the GpC
oligonucleotide failed to induce specific Abs to HSP60 or p277, the
induction of these specific Abs by the pcDNA3 vector may be linked to
the presence of the CpG motif. Thus, stimulation of the NOD immune
system with an immunostimulatory sequence alone can trigger the
production of specific autoantibodies to HSP60 and its peptide
p277.
It was conceivable that the appearance of Abs to p277 and the HSP60 Ag
reflected a polyclonal activation of IgG-secreting clones. Therefore,
we assayed the sera from the different groups of mice for Abs to
insulin, GAD, and the bacterial recombinant protein GST. Fig. 5B shows that the levels of Abs to insulin, GAD, or GST were
essentially the same among the groups. Thus, administration of pHSP60,
the pcDNA3 vector, or the CpG oligonucleotide induced specific Abs to
HSP60 and p277. This indicates that the induction of specific Abs to
HSP60 and p277 was not the result of polyclonal activation.
CpG injection inhibits NOD diabetes
To test whether administration of the CpG oligonucleotide can,
like the pcDNA3 vector, modulate the development of spontaneous
diabetes in NOD mice, we vaccinated groups of 8-wk-old female NOD mice
three times at weekly intervals and followed their glucose levels. Fig. 6 shows the cumulative incidence of
diabetes. Both untreated animals and those treated with PBS developed
the expected incidence of diabetes for NOD females; about 85% of them
were sick by the age of 6 mo. Furthermore, the incidence of diabetes
was not affected in the group of mice vaccinated with the control
oligonucleotide GpC. However, the mice vaccinated with CpG showed a
significant reduction in the incidence of diabetes. Only about 40% of
those treated with CpG (p < 0.015) were
diabetic at the age of 6 mo.
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Ab isotypes
The isotype of specific serum Abs characterizes the phenotype of
the immune response to an Ag; the Ab isotype reflects the in vivo
integration of the complex network of cytokines that regulates the
immune response. Abs of the IgG1 and IgG2b isotypes evidence a specific
Th2 response, because they are dependent on IL-4 and TGF-,
respectively (18, 19). In contrast, Abs of the IgG2a
isotype are IFN-
dependent, and they reveal the existence of a Th1
response (18, 19). Therefore, we studied the isotypes of
the Abs to p277 and HSP60 detected in DNA-vaccinated mice 14 days after
the last injection. Fig. 7 shows that the
Abs induced to HSP60 and p277 were predominantly of the IgG2b isotype
(p < 0.01 vs IgG2a levels). There was also a
slight increase in the levels of IgG1 Abs to HSP60 and p277, but this
induction was significant compared with the amount of the
IgG2a-specific Abs only in the group treated with the CpG
oligonucleotide. Furthermore, there were no differences in the isotypes
of the Abs among the pHSP60-, pcDNA3-, and CpG-treated NOD mice. Thus,
the inhibition of diabetes induced by the DNA plasmids or by the CpG
oligonucleotide in both cases was accompanied by the induction of Abs
to HSP60 and p277 of the IgG2b isotype, characteristic of a Th2-type
response.
Interestingly, there was a marked difference in the Abs induced in the
BALB/c compared with the NOD mice. The BALB/c mice made Abs to HSP60
when they were vaccinated with pHSP60 but not following immunization
with pcDNA3 (Fig. 1). Moreover, the Abs induced were mainly of the IgG1
subclass, and the BALB/c mice did not make Abs to p277. These results
indicate strain-specific differences in the cytokine networks that
regulate Ab secretion to the self-Ag HSP60.
Induction of IL-10 and IFN- secretion by the CpG oligonucleotide
To gain some insight into the cytokine effects of CpG, we assayed
the amounts of IL-10, a Th2 cytokine, and IFN-, a Th1 cytokine,
secreted by NOD spleen cells after CpG oligonucleotide stimulation in
vitro. Because different cytokines are secreted in different
physiological amounts, we included control groups of spleen cells
incubated with Con A, a prototypic T cell mitogen. As shown in Fig. 8, the CpG oligonucleotide induced both
IL-10 and IFN- production in NOD spleen cells in a dose-dependent
manner. However, compared with the amount of cytokine released in
response to Con A stimulation, the effect of CpG treatment seemed to be
relatively more effective in stimulating IL-10 than in stimulating
IFN-. CpG triggered a maximal release of IFN- of 7 ng/ml, about
one-fourth of the IFN- released by Con A. In contrast, CpG induced
the release of 1.5 ng/ml of IL-10, almost 10 times more than the amount
induced by Con A stimulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). However, we made three unexpected observations
when we used DNA treatment in NOD mice. First, the pcDNA3 plasmid,
which did not contain any sequences encoding HSP60, was as effective in
inhibiting the development of diabetes as was the pHSP60 plasmid (Figs. 2 and 3). Second, the pcDNA3 plasmid, despite the absence of HSP60,
could still induce specific effects on the autoimmunity to HSP60
intrinsic to the NOD diabetogenic process: down-regulation of T cell
proliferation and the induction of IgG2b Abs to whole HSP60 and its
critical p277 peptide. Responses to other Ags implicated in NOD
diabetes, GAD and insulin, were not detected (Figs. 4, 5, and 7).
Third, the CpG oligonucleotide by itself could essentially reproduce
the effects of the pcDNA3 plasmid on HSP60 autoimmunity and on diabetes
(
Figs. 5–7). The CpG oligonucleotide is an immunostimulatory sequence present primarily in bacteria (14, 15, 16), and our results using CpG might explain one of the mechanisms by which bacterial infections can inhibit the development of diabetes in NOD mice (20); bacterial infections may supply CpG stimulation.
It is noteworthy that the Abs to HSP60 and peptide p277 were of the
IgG2b isotype (Fig. 7). The cytokine required for the production of
IgG2b Abs is TGF-, known for its suppressive effects (18, 19). TGF- is a Th2-associated cytokine that has been shown to
protect NOD mice from diabetes (21). Although DNA
vaccination also induced HSP60- and p277-specific Abs of the IgG2a
subclass, considered to be IFN- dependent, the amount of these Abs
was significantly less than the amount of IgG2b Abs. Thus, the cytokine
balance was weighted more toward a Th2 response, suggesting that the
therapeutic effects of DNA might be related to the activation of
Th2-like T cells. Activation of Th2-like T cells was also described
when spontaneous diabetes of NOD was prevented by the administration of
the HSP60-derived peptides p12 or p277 (6, 22). Such T
cells might suppress the Th1 T cells thought to be involved in the
damage to the -cells (22). Further studies are needed
to directly confirm the involvement of particular cytokines.
The origin of the Abs to HSP60 and p277 in mice protected from diabetes
by pcDNA3 or treatment with the CpG oligonucleotide or induced by the
CpG oligonucleotide (Fig. 5A) remains to be clarified.
Clearly, this effect is strain specific; BALB/c mice did not produce
these Abs when injected with pcDNA3 (Fig. 1). NOD mice seem to manifest
a spontaneous autoimmune response to HSP60 and p277, which is depicted
in Fig. 4. Immunity to HSP60 and p277 manifests as a peak of T cell
reactivity before the onset of the disease (13, 23).
Months after the onset of overt diabetes, Abs to HSP60 and p277 can be
detected (3). After DNA treatment the T cell proliferative
response was diminished and replaced by the production of Abs, mostly
IgG2b. This suggests that the pre-existing autoimmune response,
spontaneously arising in NOD mice, changes its phenotype after
activation by bacterial DNA or CpG motifs, leading to the induction of
Th2-like, IgG2b Abs. Similarly, prevention of NOD diabetes by idiotypic
induction of lupus with a mAb was also associated with the induction of
specific Abs to HSP60 and p277 (3). Thus, even when the
induction of Abs to HSP60 and p277 does not result from specific
immunization, the appearance of such Abs seems to serve as an indicator
of the arrest of the diabetogenic process.
The effect of bacterial DNA on autoimmune inflammation is intriguing.
Bacterial DNA contains immunostimulatory motifs consisting of a central
unmethylated CpG dinucleotide flanked by two 5' purines and two 3'
pyrimidines (24). CpG motifs are under-represented in
mammalian genomes due to a combination of CpG suppression and CpG
methylation (25). This motif stimulates Th1 responses in
vivo (25). However, modulation of autoimmune conditions by
bacterial DNA has been already reported. Gilkeson et al. demonstrated
that immunization with bacterial DNA can modulate renal disease in
autoimmune NZB/NZW mice, while calf thymus DNA was not effective
(26). Furthermore, improvement in renal disease was
associated with the induction of Abs to glomerular Ags immediately
after immunization (26). Boccacio and her colleagues have
reported that noncoding plasmid DNA can inhibit experimental allergic
encephalomyelitis while activating IFN- in vitro (17).
How could a motif classically associated with a Th1 phenotype be
associated with the inhibition of diabetes, known to be Th2 mediated?
This paradox has been observed repeatedly in animal models of
spontaneous diabetes. PolyI:C, IFN-, IL-12, TNF-
, and IL-18, all
of which are well-known inducers or mediators of Th1 responses, were
shown to decrease insulitis and prevent diabetes (27, 28, 29, 30, 31, 32).
Furthermore, in the case of IL-18, protection was associated with
systemic activation of Th1-type immunity together with a shift to a Th2
phenotype of the cells infiltrating the islets (29).
Therefore, nonspecific stimulation of the NOD immune system, even by
Th1 inducers, is able to reset the ongoing immune response to islet Ags
and arrest the diabetogenic process. When we analyzed in vitro the
effect of the CpG oligonucleotide on NOD spleen cells, it clearly
induced IFN- and IL-10 in a dose-dependent manner (Fig. 8). However,
when the amounts of cytokine produced by CpG were compared with those
triggered by Con A stimulation, it was evident that the effect of the
CpG motif favored the release of IL-10. Perhaps the prominence of IL-10
is important in modulating the diabetogenic process. Other explanations
are possible, and more work must be performed to clarify the CpG
effect. Nevertheless, the present results encourage the study of
therapies aimed to activate pre-existing regulatory networks for the
management of IDDM (13, 33).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Prof. Irun R. Cohen, Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel.
3 Abbreviations used in this paper: NOD, nonobese diabetic; IDDM, insulin-dependent diabetes mellitus; HSP60, 60-kDa heat shock protein; GAD, glutamic acid decarboxylase.
Received for publication June 20, 2000. Accepted for publication August 30, 2000.
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M. Cohen-Sfady, G. Nussbaum, M. Pevsner-Fischer, F. Mor, P. Carmi, A. Zanin-Zhorov, O. Lider, and I. R. Cohen Heat Shock Protein 60 Activates B Cells via the TLR4-MyD88 Pathway J. Immunol., September 15, 2005; 175(6): 3594 - 3602. [Abstract] [Full Text] [PDF]
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D. Zipris, E. Lien, J. X. Xie, D. L. Greiner, J. P. Mordes, and A. A. Rossini TLR Activation Synergizes with Kilham Rat Virus Infection to Induce Diabetes in BBDR Rats J. Immunol., January 1, 2005; 174(1): 131 - 142. [Abstract] [Full Text] [PDF]
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F. J. Quintana, P. H. Hagedorn, G. Elizur, Y. Merbl, E. Domany, and I. R. Cohen Functional immunomics: Microarray analysis of IgG autoantibody repertoires predicts the future response of mice to induced diabetes PNAS, October 5, 2004; 101(suppl_2): 14615 - 14621. [Abstract] [Full Text] [PDF]
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 R. Nahum, O. Brenner, M.A. Zahalka, L. Traub, F. Quintana, and C. Moroz Blocking of the placental immune-modulatory ferritin activates Th1 type cytokines and affects placenta development, fetal growth and the pregnancy outcome Hum. Reprod., March 1, 2004; 19(3): 715 - 722. [Abstract] [Full Text] [PDF]
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D. S. Riminton, R. Kandasamy, D. Dravec, A. Basten, and A. G. Baxter Dermal Enhancement: Bacterial Products on Intact Skin Induce and Augment Organ-Specific Autoimmune Disease J. Immunol., January 1, 2004; 172(1): 302 - 309. [Abstract] [Full Text] [PDF]
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P. P. Ho, P. Fontoura, P. J. Ruiz, L. Steinman, and H. Garren An Immunomodulatory GpG Oligonucleotide for the Treatment of Autoimmunity via the Innate and Adaptive Immune Systems J. Immunol., November 1, 2003; 171(9): 4920 - 4926. [Abstract] [Full Text] [PDF]
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F. J. Quintana, P. Carmi, F. Mor, and I. R. Cohen DNA Fragments of the Human 60-kDa Heat Shock Protein (HSP60) Vaccinate Against Adjuvant Arthritis: Identification of a Regulatory HSP60 Peptide J. Immunol., October 1, 2003; 171(7): 3533 - 3541. [Abstract] [Full Text] [PDF]
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M. Pandarpurkar, L. Wilson-Fritch, S. Corvera, H. Markholst, L. Hornum, D. L. Greiner, J. P. Mordes, A. A. Rossini, and R. Bortell Ian4 is required for mitochondrial integrity and T cell survival PNAS, September 2, 2003; 100(18): 10382 - 10387. [Abstract] [Full Text] [PDF]
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C. Seifarth, S. Pop, B. Liu, C. P. Wong, and R. Tisch More Stringent Conditions of Plasmid DNA Vaccination Are Required to Protect Grafted Versus Endogenous Islets in Nonobese Diabetic Mice J. Immunol., July 1, 2003; 171(1): 469 - 476. [Abstract] [Full Text] [PDF]
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F. J. Quintana, M. Pitashny, and I. R. Cohen Experimental autoimmune myasthenia gravis in naive non-obese diabetic (NOD/LtJ) mice: susceptibility associated with natural IgG antibodies to the acetylcholine receptor Int. Immunol., January 1, 2003; 15(1): 11 - 16. [Abstract] [Full Text] [PDF]
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F. J. Quintana, P. Carmi, and I. R. Cohen DNA Vaccination with Heat Shock Protein 60 Inhibits Cyclophosphamide-Accelerated Diabetes J. Immunol., November 15, 2002; 169(10): 6030 - 6035. [Abstract] [Full Text] [PDF]
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W. Karges, K. Pechhold, S. Al Dahouk, I. Riegger, M. Rief, A. Wissmann, R. Schirmbeck, C. Barth, and B. O. Boehm Induction of Autoimmune Diabetes Through Insulin (but Not GAD65) DNA Vaccination in Nonobese Diabetic and in RIP-B7.1 Mice Diabetes, November 1, 2002; 51(11): 3237 - 3244. [Abstract] [Full Text] [PDF]
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 J.-F. Bach The Effect of Infections on Susceptibility to Autoimmune and Allergic Diseases N. Engl. J. Med., September 19, 2002; 347(12): 911 - 920. [Full Text] [PDF]
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F. J. Quintana, P. Carmi, F. Mor, and I. R. Cohen Inhibition of Adjuvant Arthritis by a DNA Vaccine Encoding Human Heat Shock Protein 60 J. Immunol., September 15, 2002; 169(6): 3422 - 3428. [Abstract] [Full Text] [PDF]
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