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Department of Pathology and Center for Clinical Immunobiology and Transplantation, McGill University, Montreal, Quebec, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA
expression was depressed, and the ratio of IFN- to IL-4 mRNA was
decreased, as determined by semiquantitative reverse-transcription PCR.
In contrast, NOD mice injected with a vector encoding the
proinflammatory cytokine IFN- developed diabetes earlier.
Intramuscular administration of cytokine-encoding plasmid vectors
proved to be an effective method of cytokine delivery in these mice,
and altered autoimmune disease expression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we focused on the potential immunosuppressive role of
TGF-ß1 in autoimmune murine diabetes. TGF-ß1 is a pleiotropic
cytokine with multiple antiinflammatory effects. It suppresses the
activity of T cells, macrophages, NK cells, and B cells, and inhibits
the expression of many proinflammatory cytokines such as IFN-,
TNF-
, IL-1, and IL-2 (4). We hypothesized that TGF-ß1 could
prevent IDDM in NOD mice, and that there would be several advantages to
delivering this cytokine by a somatic gene therapy approach.
It has been shown that i.m. injection of naked plasmid expression
vectors results in the cellular uptake of the plasmid DNA, which is
maintained episomally for prolonged periods of time within skeletal
muscle cells (5). In this study, we demonstrate that i.m. injection of
a vector encoding mouse TGF-ß1 cDNA, i.e., pCMV-TGF-ß1, results in
uptake, retention, and expression of this vector. There is detectable
vector-derived TGF-ß1 mRNA in skeletal muscle cells, as well as
increased levels of TGF-ß1 in the plasma of treated mice.
Administration of pCMV-TGF-ß1 was effective at suppressing a DTH
response, and at protecting NOD mice from insulitis and diabetes. There
is a decreased expression of IL-12 and IFN- mRNA in the pancreas of
protected mice. In contrast, administration of an IFN--encoding
vector accelerated disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female NOD mice (8–10 wk) were purchased from Taconic Farms (Germantown, NY), and female BALB/c mice (substrain AnNCrlBR, 4–6 wk) were purchased from Charles River Canada (St-Constant, Quebec, Canada). Mice were kept in a pathogen-free facility. These NOD mice exhibit mild insulitis as early as 4 wk of age and become diabetic starting at about 12 wk of age.
Expression vector construction and TGF-ß1 assays
The mouse TGF-ß1 (mTGF-ß1) cDNA was produced by RT-PCR from
Con A-stimulated BALB/c splenocytes and cloned into compatible enzyme
restriction sites of pCI-neo (Promega, Madison, WI) to generate
pCMV-TGF-ß1. The mTGF-ß1 cDNA is under the transcriptional control
of a CMV immediate-early enhancer/promoter, and downstream of a
chimeric intron. pCMV-TGF-ß1 encodes the latent form of mTGF-ß1,
and expression was confirmed using a TGF-ß1 ELISA (R&D Systems,
Minneapolis, MN) in supernatants collected from transiently transfected
COS-7 cells. The TGF-ß1 ELISA only detects active TGF-ß1. The
latent TGF-ß1 was activated to its biologically active form by
acidification for 10 min, and its bioactivity was confirmed by the
CCL64 mink cell line proliferation assay. Mouse IFN- cDNA was
subcloned into pCI-neo, as described above, to generate pCMV-IFN-.
pCI-neo, henceforth referred to as pCMV-null, was used as control
vector in all of the experiments.
Plasmid DNA preparation
Large-scale plasmid DNA preparations were produced by the
alkaline lysis method using a Qiagen giga kit (Qiagen, Santa Clarita,
CA). All plasmid preparations for i.m. injections were resuspended in
sterile 0.85% saline. Spectrophotometric analysis revealed 260/280 nm
ratios 
1.80. Purity of DNA preparations and conformations was
confirmed on a 1% agarose gel.
Determination of plasma TGF-ß1 levels
To minimize the activation of platelets and subsequent release of endogenous TGF-ß1, platelet-poor plasma was obtained as follows: whole blood was mixed with a 1.5% EDTA solution, mixed thoroughly, and put on ice immediately after blood collection. The blood/EDTA mixture was then layered gently on 20% sucrose and centrifuged for 30 min at 12,000 x g. The upper two-thirds of the upper phase (platelet-poor plasma fraction) were collected without disturbing the interface. Once separated, the platelet-poor plasma was frozen at -80°C until assayed for TGF-ß1 by ELISA. Statistical analysis was performed by Student’s t test.
Induction and measurement of DTH reaction in mice
On day 1, BALB/c mice were immunized with 200 µg of OVA in CFA, or with CFA alone. On day 6, mice were injected with OVA in PBS in the right footpad. DTH responses were measured, with calipers, as the increase in footpad thickness 24, 36, and 48 h after OVA Ag recall immunization. Statistical analysis was performed with the Student’s t test.
Intramuscular injection of plasmid DNA
Intramuscular injections of plasmid DNA were done as described
(6). Briefly, mice were anesthetized by i.p. injection with xylazine
(10 mg/kg) and ketamine (200 mg/kg). The rectus femoris (RF) and
tibialis anterior (TA) muscles of each mouse were injected with a
0.5-cc sterile 29G1/2 insulin syringe, fitted with a plastic collar to
limit needle penetration to 2 mm. Mice received 100 µg of
pCMV-TGF-ß1 in 50 µl of sterile saline in each RF or TA muscle, for
a total of 400 µg of plasmid DNA in each treatment session, unless
stated otherwise. Control mice received equivalent amounts of pCMV-null
control vector in each muscle group. A vector encoding mouse IFN-,
pCMV-IFN-, was administered in a similar fashion.
Induction and diagnosis of diabetes
Cyclophosphamide (CYP; Sigma, St. Louis, MO), which accelerates the onset of diabetes in NOD mice (7), was administered i.p. twice, 14 days apart, at a dose of 200 mg/kg in PBS to 8- to 10-wk-old female NOD mice. Diabetes was diagnosed by regular urinary glucose analysis and confirmed by blood glucose determination. Mice were considered diabetic when sequential blood glucose measurements were shown to be equal to, or above 16.7 mmol/L (300 mg/dl), as determined by the Accu-ChekIII glucometer (Boehringer Mannheim, Indianapolis, IN).
Statistical analysis was done using the SAS software (version 6.12) for Windows 95. The incidence of diabetes was plotted using the Kaplan-Meier method (nonparametric cumulated survival plot). The statistical comparison between the curves obtained was performed using the Wilcoxon log rank test.
Histologic analysis
The pancreas was excised immediately after CO2
asphyxiation and fixed in 10% buffered Formalin, and hematoxylin and
eosin-stained histologic slides were prepared. Insulitis was graded as
follows: grade 0, normal islet totally free of any periislet
mononuclear cells; grade 1, focal periislet lymphocytic infiltration
<25% of islet circumference; grade 2, periislet lymphocytic
infiltration >25% of islet circumference; grade 3, mild insulitis,
intraislet infiltration with good retention of islet cell morphology;
grade 4, severe insulitis with significant destruction of ß-islet
cells. Three randomly obtained levels of pancreas were analyzed in
double-blind fashion by two observers. Statistical analysis was
performed with the 
2 test.
PCR and RT-PCR analysis
Mice were killed and their TA muscles were excised, immediately
frozen in liquid nitrogen, and stored at -80°C. Total genomic DNA
was isolated from thawed muscle specimens, as described (8). The PCR
reactions were performed in a 50 µl reaction vol containing 2.5 µl
genomic DNA, 10 mM Tris-HC1, pH 8, 50 mM KC1, 2 mM dNTP, 5 mM
MgC12, 2 µM of each primer, and 1.5 U Taq DNA
polymerase. The primer sequences amplifying vector-encoded TGF-ß1
were 5'-AGAGAAGAACTGCTGTGTGCGGCAG-3' (sense) and
5'-CGCTTCCCTTTAGTGAGGGTTAATG-3' (antisense). The TGF-ß1 primer set
amplified a TGF-ß1 product from pCMV-TGF-ß1 DNA, or cDNA derived
from that vector, but not from genomic DNA or endogenous TGF-ß1 cDNA.
PCR cycling conditions were as follows: one cycle at 94°C; 40 cycles
at 94°C for 1 min, at 55°C for 2 min, and 72°C for 2 min; and one
final extension cycle at 72°C for 10 min. The PCR amplifiers were
analyzed on a 1.5% agarose gel containing 0.5 µg/ml ethidium
bromide. The TGF-ß1 mRNA in treated muscle was detected by
RT-PCR. Total RNA was extracted from entire TA muscles, as described
(8), and reverse transcribed with the Superscript preamplification
system (Life Technologies, Gaithersburg, MD). A total of 2 µl
of the reverse-transcription reaction was used for PCR amplification
using the above-mentioned vector-specific primers for pCMV-TGF-ß1, or
G3PDH primers (sense, 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3', and
antisense, 5'-CATGTAGGCCATGAGGTCCACCAC-3'). Optimal amplification
conditions were conducted for 40 cycles and amplifiers were labeled
with [-32P]dCTP (5 µCi) (ICN, Mississauga, ON). The
PCR products were analyzed on 1.5% agarose gel containing 0.5 µg/ml
ethidium bromide, transferred onto Hybond-N+ nylon membrane
(Amersham Canada, Oakville, ON), and either exposed to autoradiographic
film or subjected to phosphor imager analysis.
For intrapancreatic cytokine mRNA analysis, total RNA was isolated from
snap-frozen pancreas and quantification of specific cytokine gene
expression was performed by RT-PCR, as described above. For this
purpose, specific primers were used in PCR for IL-12 p40 (9), IL-4
(10), IFN- (10), and ß-actin (11). Analysis of cytokine PCR
products was performed as previously described (11). Briefly, PCR
reactions were terminated in the linear portion of the amplification
reaction (which extended up to 35 cycles). The 32P-labeled
PCR products were analyzed on a 2% agarose gel, transferred onto a
nylon membrane, and subjected to phosphor imager analysis.
Semiquantitative mRNA analysis was performed by calculating relative
quantities of RT-PCR signals for each cytokine, normalized to the
ß-actin signal of each sample. The ratio of IFN- to IL-4 mRNA (for
a mouse) was derived after normalization of these cytokines with their
ß-actin signal, as described above: IFN-/IL-4 ratio =
[(IFN-/ß-acton ratio)/(IL-4/ß-actin ratio)].
In all of these experiments, specificity of PCR products was confirmed by restriction enzyme analysis. Statistical analysis was performed with Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether plasmid DNA is successfully retained and
expressed by treated muscle tissue, each TA muscle was injected with
100 µg of pCMV-TGF-ß1, followed by total DNA or RNA extraction, and
PCR or RT-PCR analysis, respectively. We found that 14 days following
i.m. injection of pCMV-TGF-ß1, a 348-bp amplifier, specific for this
vector, could readily be detected by PCR analysis from all treated
muscle samples (Fig. 1
A), and
was undetectable in null-vector-treated mice (Fig. 1B).
Furthermore, RT-PCR analysis of RNA samples extracted from these
muscles revealed the presence of vector-derived TGF-ß1 transcripts
(Fig. 1C), which were absent in pCMV-null-treated mice (Fig. 1D). No product was identified from RNA preparations when
reverse transcription was omitted, and the PCR primers could not
amplify endogenous TGF-ß1 from either genomic DNA or cDNA (not
shown).
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To establish the optimal dose of plasmid DNA, increasing amounts
of pCMV-TGF-ß1 were injected at different muscle sites, and TGF-ß1
levels in platelet-poor plasma were measured by TGF-ß1 ELISA, 75
h postinjection. We found that vector expression, as determined by
plasma TGF-ß1 levels, increases with the number of injection sites
and amount of DNA injected per site, up to 100 µg of DNA per
injection site (data not shown). To determine whether the elevation of
TGF-ß1 levels was sustained, female NOD mice were injected with a
total of 200 µg of plasmid DNA (100 µg in each TA muscle). TGF-ß1
levels of 9.2 ± 0.4 ng/ml were observed at 36 h
postinjection, compared with 2.6 ± 0.4 ng/ml in pCMV-null-treated
mice (Fig. 2). These levels decreased to
a mean plasma level of 4.3 ± 0.3 ng/ml on day 14
(p < 0.05 at 36 h and 14 days versus
null-vector-treated mice). At day 35, TGF-ß1 was still slightly
higher in the pCMV-TGF-ß1-treated mice, but this was no longer
statistically significant. TGF-ß1 plasma levels in age-matched,
untreated NOD mice were 2.3 ± 0.5 ng/ml, a value statistically
different from the pCMV-TGF-ß1-treated mice at 36 h and day 14
(p < 0.05), but not statistically different
from pCMV-null treated mice at any time point. In all cases, active
TGF-ß1 was either absent or present at undetectable levels in the
plasma of treated mice (not shown).
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To examine whether i.m. injections of plasmid DNA result in
sufficient production of TGF-ß1 to exert biologic effects, we used a
DTH model. DTH responses were assessed by footpad swelling 24 to
48 h following recall immunization with OVA in female BALB/c mice.
We found that pCMV-TGF-ß1-treated mice had significantly suppressed
DTH responses (Fig. 3). These mice had a
sevenfold reduction in DTH responses observed at 36 h, versus
pCMV-null-treated mice, consistent with the in vivo production and
activation of TGF-ß1.
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To determine whether administration of pCMV-TGF-ß1 could prevent
autoimmune disease expression, we administered the TGF-ß1 plasmid
expression vector to adult NOD female mice. The autoimmune basis of
this disease is well established (12). To accelerate disease
expression, female NOD mice were injected with CYP, as described (13).
CYP has been reported to accelerate disease in NOD mice by enhancing
intraislet production of IFN- and other inflammatory mediators (14).
Briefly, CYP was administered i.p., on days 3 and 16, at a dose of 200
mg/kg. Animals received a total of 100 µg of pCMV-TGF-ß1 in sterile
saline in each RF and TA muscle for a total of 400 µg of plasmid DNA,
48 h before each CYP injection. Control animals received
equivalent amounts of pCMV-null control vector in each muscle. The
incidence of IDDM was evaluated by sequential measurements of blood
glucose levels.
Administration of pCMV-TGF-ß1 significantly reduced the incidence of
diabetes in NOD female mice (Fig. 4A). The first case of
diabetes occurred 14 days later in the pCMV-TGF-ß1 group, compared
with control group. By day 32 of the experiment, the incidence of
diabetes was four times higher in pCMV-null-treated mice compared with
mice receiving pCMV-TGF-ß1 (p < 0.001). In
diabetic mice, the hyperglycemia (300 mg/dl) was maintained 2 wk or
more following onset of disease (data not shown).
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administration
Diabetes occurred earlier in pCMV-IFN--treated mice compared
with control mice (Fig. 4
A, p = 0.05, versus
null-vector-treated mice). The pCMV-mIFN--treated mice had increased
serum levels of IFN- (up to 200 pg/ml, as determined by ELISA),
while this cytokine was undetectable in the serum of control mice (data
not shown).
Protective effect of pCMV-TGF-ß1 treatment on the natural course of diabetes
To determine an effect on the natural course of disease, we
injected female NOD mice (9–11 wk) with pCMV-TGF-ß1 in the absence
of CYP (Fig. 4B). Under these conditions, the incidence of
diabetes was again significantly reduced, to approximately 50% of
control values over the course of several weeks
(p < 0.002). Thus, TGF-ß1-mediated
protection is not a feature unique to the CYP-accelerated diabetes
model.
Protection from insulitis in pCMV-TGF-ß1-treated NOD mice
Insulitis was graded histologically based on mononuclear cell
infiltration of pancreatic islets. In Figure 5, we compare nondiabetic mice necropsied
before the development of overt diabetes. In CYP-accelerated disease
(Fig. 5A), administration of pCMV-TGF-ß1 reduced the mean
insulitis score from 2.91 in control mice to 1.92 in treated mice
(p < 0.001). Protection was also observed in
natural course disease (Fig. 5B), in which the mean
insulitis score was 3.44 in control mice and 2.25 in treated mice
(p < 0.01). Thus, pCMV-TGF-ß1 treatment
induced a shift to lower grade lesions, including increased number of
normal islets (Fig. 5, A and B). This shift was
more marked in CYP-induced disease than in natural course disease, but
was highly significant in both cases.
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mRNA expression in
pCMV-TGF-ß1-treated NOD mice
Analysis of intrapancreatic cytokine expression patterns in
CYP-treated mice showed marked differences between the prediabetic
pCMV-TGF-ß1-treated and control mice. Compared with pCMV-null control
mice, pCMV-TGF-ß1-treated mice had lower levels of both IFN-
(p = 0.002) and IL-12
(p < 0.001) mRNA (Fig. 6A). Mean IL-4 mRNA levels
were not altered significantly by pCMV-TGF-ß1 administration (a
slight decrease versus control mice was not statistically significant)
(Fig. 6A). Consequently, there was a considerable decline of
the ratio of IFN- to IL-4 mRNA levels (p <
0.05) (Fig. 6B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we demonstrate that i.m. injection of plasmid DNA-encoding latent TGF-ß1 (pCMV-TGF-ß1) results in uptake, retention, and expression of this vector by muscle cells. As demonstrated by RT-PCR, there is detectable vector-derived TGF-ß1 mRNA in skeletal muscle cells, which is not seen in null-vector-treated muscles. Moreover, plasma samples collected from pCMV-TGF-ß1-treated mice show significantly elevated levels of TGF-ß1 for well over 2 wk. Over that time period, the TGF-ß1 levels were two- to fourfold higher than those of control mice.
We found that DTH responses to OVA are markedly suppressed in pCMV-TGF-ß1-treated mice. Thus, it is apparent that sufficient quantities of TGF-ß1 are produced to exert an immunosuppressive effect. Moreover, the production of TGF-ß1 protects NOD mice from an autoimmune disease.
NOD mice spontaneously develop an autoimmune form of insulitis, with
destruction of ß-islet cells (reviewed in 12 . Insulitis is the
precursor lesion, leading to diabetes when it is sufficiently severe.
This disease is clearly T cell and macrophage dependent, although the
mechanisms of islet cell destruction is not fully elucidated. Islet
cells may be killed by infiltrating CTLs, NK cells, and macrophages
(12). Proinflammatory cytokines (IL-1, IFN-, TNF-), as well as
nitric oxide (NO), are toxic to islet cells (14, 21). Th1-dependent
immunity appears to be a key pathogenic factor, since, for example,
administration of IL-12 enhances Th1 reactivity to islet cell Ags and
rapidly induces diabetes (22). IL-12 and IFN- are produced locally
in inflamed islets, and insulitis can be induced by adoptive transfer
of islet-reactive Th1 clones (23). The administration of CYP to NOD
mice stimulates intraislet production of IFN- and other inflammatory
mediators, and results in rapid onset of diabetes (14).
We found that administration of pCMV-TGF-ß1 considerably reduced incidence of diabetes in NOD mice. In CYP-induced diabetes, there was a fourfold reduction in incidence (day 32 of the experiment). In non-CYP-treated mice (natural course), treatment delayed the appearance of the first case of diabetes, and subsequently reduced the incidence of disease by approximately 50% over the course of several weeks. In the latter mice, treatment was begun at 9 to 11 wk, i.e., at a time when insulitis is already apparent, suggesting that an ongoing autoimmune response was suppressed.
pCMV-TGF-ß1-treated mice had low insulitis scores, with markedly
increased numbers of normal, or islets with mononuclear cell
infiltrates limited to the periislet area (periinsulitis). In
TGF-ß1-treated mice following CYP administration, analysis of
pancreatic cytokine mRNA expression by semiquantitative RT-PCR revealed
depressed IL-12 and IFN- levels. IL-4 levels were not significantly
altered. Consequently, the ratio of IFN- to IL-4 mRNA was reduced by
TGF-ß1 plasmid DNA therapy. Protection from insulitis was somewhat
greater in CYP-accelerated than in natural course disease, but was
statistically significant in both cases. Taken together, our results
reveal that TGF-ß1 plasmid therapy had an antiinflammatory or
immunosuppressive effect. As expected, mice that were protected from
insulitis were also protected from diabetes.
The immunoregulatory influence of TGF-ß1 has been studied in many in vivo models, including experimental allergic encephalomyelitis (24), collagen-induced arthritis (25), and allograft rejections (26). In all of these instances, microgram amounts of TGF-ß1 were administered, over a period of a few days, to achieve a significant suppression of autoimmune responses. Since we could suppress DTH and prevent autoimmune disease expression with i.m. injections of pCMV-ß1, it appears that we are achieving results equivalent to administration of microgram quantities of TGF-ß1 protein.
TGF-ß1 may act at multiple levels to block inflammatory reactions
and/or prevent autoimmune disease. The potent immunosuppressive effects
of TGF-ß1 were clearly demonstrated in the TGF-ß1 knockout mice
that died at an early age with a multiorgan inflammatory syndrome (27).
The absence of TGF-ß1 was associated with increased production of
inflammatory cytokines such as IFN- and TNF-, and an increased
number of activated immune cells in peripheral lymphoid organs.
Two recent studies (9, 28) demonstrate that TGF-ß (ß1 or ß2) can
modulate macrophage activity in a way that favors Th2 over Th1
differentiation. Our results are consistent with these observations,
since with TGF-ß1 therapy we see suppression of the type 1
proinflammatory cytokines IL-12 and IFN-, with minimal alteration of
IL-4 gene expression. Based on published data, the regulatory activity
of TGF-ß1 may result from a direct action on macrophages or may be a
consequence of decreased Th1 reactivity. In fact, this cytokine could
be blocking several steps of an immune reaction, including Ag
processing/presentation by APCs (29), activation/differentiation of Th1
cells (30), production of inflammatory cytokines and NO (30, 31), and
activation of effector cells (CTLs, macrophages, NK cells) (32).
TGF-ß1 blocks JAK-STAT signaling in T cells by preventing tyrosine
phosphorylation and activation of Jak-1 and STAT-5 (33). It inhibits
IL-2R expression, and can also induce apoptosis in T cells (33). These
mechanisms are not mutually exclusive, and it would be difficult to
ascertain their relative importance in vivo in NOD mice. Interestingly,
the increased number of normal islets in TGF-ß1-treated mice suggests
that either fewer islet-reactive T cells are activated or that their
migration to islets of Langerhans is impeded. The site of activation of
latent TGF-ß1 is unclear, as discussed below.
Previously, others found that i.m. injection of TGF-ß1 expression vectors was therapeutically effective in models of inflammatory bowel disease (34) and systemic lupus erythematosus (35). These investigators administered plasmid vectors with a Rous sarcoma virus enhancer/promoter (compared with a CMV enhancer/promoter in our study), and either did not report an increase in plasma TGF-ß1 (34), or observed a smaller increase than in our experiments (35). It appears that we achieved a higher level of TGF-ß1 production, possibly because the CMV enhancer/promoter of our vector generates a higher transcriptional activity (5, 8).
Adoptive transfer of TGF-ß1-producing islet-reactive CD4+ T cells prevents diabetes in NOD mice (36). However, transgenic NOD mice producing active TGF-ß1 in their islets (rat insulin promoter) developed insulitis and marked fibrosis in the pancreas (37). Immunoprotection may depend on the TGF-ß1 levels achieved locally or systemically, and/or the state of TGF-ß1 activation.
The ability of TGF-ß1 to induce fibrosis and extracellular matrix formation (38) is a potential concern. In mice treated with pCMV-TGF-ß1, we did not observe fibrosis or inflammation in muscles, kidneys, lungs, liver, heart, or pancreas. We speculate that the latent TGF-ß1 is not activated at the site of plasmid administration, since active TGF-ß1 was not detectable in plasma. Others have shown (26) that administration of latent TGF-ß1 is probably an advantageous feature, since it may become biologically active only at distant inflammatory sites, through the action of macrophages, low pH, or other factors (26).
As mentioned above, IFN- may contribute to islet cell injury, since
it has direct toxic effects on islet cells, and may also act by
activating macrophages and stimulating NO production. In accordance
with this view, disease is delayed in IFN- gene knockout NOD mice
(39), and prevented by anti-IFN- mAb treatment (40). To confirm
the detrimental effects of IFN-, as well as the efficacy of our gene
delivery method, we administered an IFN--expressing vector
(pCMV-IFN-) to CYP-treated NOD mice. The serum levels of IFN-
were increased in pCMV-IFN--treated mice (up to 200 pg/ml), and this
was sufficient to induce an earlier onset of diabetes. Acceleration of
this disease by administration of IFN- had not been reported before.
To our knowledge, we are the first to demonstrate that IDDM can be prevented by cytokine i.m. somatic gene therapy, and to demonstrate that TGF-ß1 is therapeutically effective in this disease. Clinically, the use of cytokines has been limited by their short t1/2 and the necessity to administer relatively large quantities (often in boluses) of recombinant proteins, with considerable associated toxicity (41). Somatic cytokine gene therapy has the potential of circumventing these problems by minimizing the need for frequent protein injections, producing more constant blood levels, reducing side effects, and increasing therapeutic efficacy. Furthermore, direct DNA injection in skeletal muscle appears to be safe and is technically simple. Unlike some viral vectors, the plasmid vectors are maintained episomally (5), minimizing the risk of genomic insertional mutagenesis. Moreover, the use of plasmid vectors eliminates the possibility of immune responses to viral Ags.
Various nonviral means of introducing DNA into cells have been developed. Although cationic liposomes usually enhance DNA uptake by cells, i.v. injection of DNA/liposome complexes has met with limited success. In that case, the cells of several organs are transfected (particularly endothelial cells) (42, 43), but the persistence of gene expression is usually much less than after i.m. delivery (43; and our unpublished observations). The potential toxicity of liposomes must also be considered (43). Thus, the muscle cell is an excellent target for gene therapy, and in these cells injection of naked plasmid DNA has proven to be an effective method.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gérald J. Prud’homme, Department of Pathology, McGill University, 3775 University Street, Montreal, Quebec, Canada, H3A 2B4. E-mail address:
3 Abbreviations used in this paper: NOD, nonobese diabetic; IDDM, insulin-dependent diabetes mellitus; CYP, cyclophosphamide; DTH, delayed-type hypersensitivity; mTGF, mouse transforming growth factor; NO, nitric oxide; RF, rectus femoris; TA, tibialis anterior.
Received for publication October 14, 1997. Accepted for publication June 15, 1998.
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 C. Tian, M. J. I. Ansari, J. Paez-Cortez, J. Bagley, J. Godwin, M. Donnarumma, M. H. Sayegh, and J. Iacomini Induction of Robust Diabetes Resistance and Prevention of Recurrent Type 1 Diabetes Following Islet Transplantation by Gene Therapy J. Immunol., November 15, 2007; 179(10): 6762 - 6769. [Abstract] [Full Text] [PDF]
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 Y. Gao, W.-P. Qian, K. Dark, G. Toraldo, A. S. P. Lin, R. E. Guldberg, R. A. Flavell, M. N. Weitzmann, and R. Pacifici Estrogen prevents bone loss through transforming growth factor {beta} signaling in T cells PNAS, November 23, 2004; 101(47): 16618 - 16623. [Abstract] [Full Text] [PDF]
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C. Tikellis, P. J. Wookey, R. Candido, S. Andrikopoulos, M. C. Thomas, and M. E. Cooper Improved Islet Morphology after Blockade of the Renin- Angiotensin System in the ZDF Rat Diabetes, April 1, 2004; 53(4): 989 - 997. [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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 A. Olivieri, S. De Angelis, V. Vaccari, H. Valensise, F. Magnani, M. A. Stazi, R. Cotichini, E. Gilardi, V. Cordeddu, M. Sorcini, et al. Postpartum Thyroiditis Is Associated with Fluctuations in Transforming Growth Factor-{beta}1 Serum Levels J. Clin. Endocrinol. Metab., March 1, 2003; 88(3): 1280 - 1284. [Abstract] [Full Text] [PDF]
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 H Schotte, P Willeke, S Rust, G Assmann, W Domschke, M Gaubitz, and B Schluter The transforming growth factor-{beta}1 gene polymorphism (G915C) is not associated with systemic lupus erythematosus Lupus, February 1, 2003; 12(2): 86 - 92. [Abstract] [PDF]
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H. Braley-Mullen, K. Chen, Y. Wei, and S. Yu Role of TGF{beta} in Development of Spontaneous Autoimmune Thyroiditis in NOD.H-2h4 Mice J. Immunol., December 15, 2001; 167(12): 7111 - 7118. [Abstract] [Full Text] [PDF]
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G. L. Costa, M. R. Sandora, A. Nakajima, E. V. Nguyen, C. Taylor-Edwards, A. J. Slavin, C. H. Contag, C. G. Fathman, and J. M. Benson Adoptive Immunotherapy of Experimental Autoimmune Encephalomyelitis Via T Cell Delivery of the IL-12 p40 Subunit J. Immunol., August 15, 2001; 167(4): 2379 - 2387. [Abstract] [Full Text] [PDF]
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D. J. Weaver Jr., B. Liu, and R. Tisch Plasmid DNAs Encoding Insulin and Glutamic Acid Decarboxylase 65 Have Distinct Effects on the Progression of Autoimmune Diabetes in Nonobese Diabetic Mice J. Immunol., July 1, 2001; 167(1): 586 - 592. [Abstract] [Full Text] [PDF]
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A. M. Yamamoto, Y. Chernajovsky, F. Lepault, O. Podhajcer, M. Feldmann, J.-F. Bach, and L. Chatenoud The Activity of Immunoregulatory T Cells Mediating Active Tolerance Is Potentiated in Nonobese Diabetic Mice by an IL-4-Based Retroviral Gene Therapy J. Immunol., April 15, 2001; 166(8): 4973 - 4980. [Abstract] [Full Text] [PDF]
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R. Tisch, B. Wang, D. J. Weaver, B. Liu, T. Bui, J. Arthos, and D. V. Serreze Antigen-Specific Mediated Suppression of {{beta}} Cell Autoimmunity by Plasmid DNA Vaccination J. Immunol., February 1, 2001; 166(3): 2122 - 2132. [Abstract] [Full Text] [PDF]
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D. V. Serreze, H. D. Chapman, C. M. Post, E. A. Johnson, W. L. Suarez-Pinzon, and A. Rabinovitch Th1 to Th2 Cytokine Shifts in Nonobese Diabetic Mice: Sometimes an Outcome, Rather Than the Cause, of Diabetes Resistance Elicited by Immunostimulation J. Immunol., January 15, 2001; 166(2): 1352 - 1359. [Abstract] [Full Text] [PDF]
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E. Dahlen, G. Hedlund, and K. Dawe Low CD86 Expression in the Nonobese Diabetic Mouse Results in the Impairment of Both T Cell Activation and CTLA-4 Up-Regulation J. Immunol., March 1, 2000; 164(5): 2444 - 2456. [Abstract] [Full Text] [PDF]
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H. M. Horton, D. Anderson, P. Hernandez, K. M. Barnhart, J. A. Norman, and S. E. Parker A gene therapy for cancer using intramuscular injection of plasmid DNA encoding interferon alpha PNAS, February 16, 1999; 96(4): 1553 - 1558. [Abstract] [Full Text] [PDF]
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