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Department of Biochemistry, University of Wisconsin-Madison, College of Agricultural and Life Sciences, Madison, WI 53706
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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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We have recently provided strong experimental evidence that
1,25-dihydroxyvitamin D3
(1,25-(OH)2D3), the functional metabolite of
vitamin D3, can greatly reduce or eliminate the incidence
and severity of experimental autoimmune encephalomyelitis (EAE), an
animal model of MS (7). EAE is mediated by CD4+ T cells,
and more specifically Th1 cells that recognize proteins in the CNS (8).
Th1 cells secreting IFN-
and TNF-
are associated with EAE in mice
(9, 10) and with MS in humans (11). Neutralization of TNF in vivo
decreases the severity of EAE in mice (12). Interestingly,
neutralization of IFN- in vivo and the use of IFN- knockout mice
suggest a beneficial role for IFN- in EAE (13, 14). A number of T
cell products and the development of Th2 cells specific for CNS
proteins are associated with the suppression of EAE in mice (15). In
particular, TGF-ß1 treatment of mice exhibiting signs of EAE has been
shown to be beneficial (16). Conversely, neutralization of TGF-ß1 in
vivo increases the severity of EAE (17). Finally, based on work with
EAE, TNF- inhibitors and exogenous TGF-ß1 have been developed as
new immunotherapies for MS (18).
There are at least two possible mechanisms whereby vitamin D might
prevent or decrease the severity of EAE. The functional metabolite or
hormonal form of vitamin D, 1,25-(OH)2D3, has
been shown to inhibit the in vitro proliferation of T cells and to
decrease the in vitro production of two Th1 cytokines, IFN- and
TNF- (19, 20, 21). The first possibility is that
1,25-(OH)2D3 is negatively regulating
encephalitogenic T cells and the cytokines they produce. A second
possibility is that 1,25-(OH)2D3 is positively
regulating anti-encephalitogenic cells and the cytokines they
produce. These potential mechanisms are not mutually exclusive.
Experiments are described below that were designed to probe the
immunobiologic mechanism(s) underlying the treatment and prevention of
EAE by 1,25-(OH)2D3. We measured four cytokines
pivotal for the outcome of EAE: IFN-, TNF-, IL-4, and TGF-ß1.
Our results support the hypothesis that
1,25-(OH)2D3 acts primarily by increasing the
anti-inflammatory cytokines IL-4 and TGF-ß1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The B10.PL mice were produced in our colony using breeding pairs obtained from The Jackson Laboratory (Bar Harbor, ME). During this time, the mice were maintained on Purina Chow that provides abundant amounts of vitamin D. At 6 to 7 wk of age, male and female mice were fed experimental purified diets described previously (7) that contained no vitamin D. Although the diets do not contain vitamin D, the mice were housed in rooms with fluorescent light that catalyzes vitamin D production in skin. Therefore, the mice were not vitamin D deficient. The amount of calcium in the diet was uniformly 0.25%. Mice were maintained for 1 wk on this experimental diet before use in experiments. Experiments used no less than 5 and up to 10 mice in each treatment group. The complete experiment with all time points were repeated at least two times but as many as seven times.
EAE induction
EAE was induced exactly as described (7). Briefly, mice were immunized s.c. with 400 µg of guinea pig myelin basic protein (MBP) in CFA. In addition, on the day of immunization and 2 days later the mice received an i.p. injection with 200 ng of pertussis toxin (List Biological Laboratories, Campbell, CA) in saline.
EAE severity was scored daily by two blinded individuals using the following scoring system: 0 = no symptoms; 1 = limp tail; 2 = paraparesis; 3 = hind limb paralysis; 4 = fore and hind limb paralysis; and 5 = moribund. Intermediate scores of ±0.5 were used for mice with symptoms in between any two scores. Twenty-five to thirty percent of control B10.PL mice developed severe EAE and were sacrificed. These animals were scored as 5 on the day they were sacrificed. The future EAE scores do not include scores from sacrificed mice.
1,25-(OH)2D3 treatment
For the first experimental design, the 1,25-(OH)2D3 treatment was started the day before EAE induction. Females were fed 1,25-(OH)2D3 at 50 ng/day and the males were fed 1,25-(OH)2D3 at 200 ng/day. These doses of 1,25-(OH)2D3 were found previously to be the minimum needed to prevent EAE completely (Ref. 7, and M. T. Cantorna, J. Humpal-Winter, and H. F. DeLuca, unpublished observations). A second experimental design was to start 1,25-(OH)2D3 treatment when the tails of immunized mice first became limp (first signs of EAE, about day 7). One group of mice was treated with an i.p. injection of 1,25-(OH)2D3 (300 ng) in 0.1 ml of propylene glycol, and the other group of mice was injected with propylene glycol alone (controls). Both male and female mice injected with 1,25-(OH)2D3 were maintained on the experimental diet to which 1,25-(OH)2D3 was added to provide 50 ng per mouse daily. Control animals were provided the experimental diet throughout. A third experimental design was to allow mice to develop severe EAE (severity score of 2.5). Mice with EAE severity scores of 2.5 were randomly placed into two groups. One group of mice was treated with an i.p. injection of 1,25-(OH)2D3 (300 ng) in 0.1 ml of propylene glycol, and the other group of mice was injected with propylene glycol alone (controls). Some of the 1,25-(OH)2D3-injected and control-injected mice were used 24 h later for cytokine transcript analyses. The others were maintained on the experiment diet (controls) or on the same diet supplemented with 1,25-(OH)2D3 to provide 50 ng per mouse daily.
Cell cultures
Axillary, brachial, and inguinal lymph nodes (LN) were collected from control- and 1,25-(OH)2D3-treated mice at days 3, 7, 10, 14, and 21 postimmunization. These LNs were chosen because they were enlarged and drained the site of immunization. Collected LNs were disrupted manually using a 23-gauge needle and a pair of forceps. Total cell numbers in the LNs were determined by counting the number of lymphocytes from control- and 1,25-(OH)2D3-treated mice and dividing by the number of mice in the group. Flow cytometry of fluorescent-labeled cell populations (Thy-1, class II, CD4, and CD8) was done on LN cells from control- and 1,25-(OH)2D3-treated mice. For cytokine PCR analysis, LN cells were saved for total cellular RNA isolation. For cytokine protein determination, equal numbers of cells from control- and 1,25-(OH)2D3-treated mice were cultured in HL-1 serum-free medium (BioWhittaker, Walkerville, MD) supplemented with 2-ME (50 µM), glutamine (2 mM), penicillin (10 U/ml), and streptomycin (10 µg/ml). To stimulate cytokine production, lymphocytes were restimulated with 25 µg/ml of MBP for 24 to 72 h. Nonrestimulated LN cells did not produce cytokines. Supernatants were harvested and assayed for cytokine production using cytokine-specific ELISAs.
Isolation of spinal cord and brain samples
Groups of five to eight control and five to eight
1,25-(OH)2D3-treated mice were sacrificed
and perfused with 10 to 15 ml of sterile saline. The treatments were
started the day before immunization and both groups of mice were
sacrificed when the controls reached EAE severity scores of 
2.5. The
spinal cords were either saved for histopathology or the spinal cords
and brains were pooled and manually homogenized in saline using a
Dounce homogenizer. The debris was allowed to settle and the cells were
resuspended in a 70% Percoll (Pharmacia, Piscataway, NJ) solution. The
cell/Percoll suspension was overlaid with a 30% Percoll solution and
spun for 15 min at 500 x g. Lymphocytes were collected
from the 30/70% interface (22, 23). The lymphocytes were washed twice
and relayered onto Histopaque 1083 (Sigma, St. Louis, MO) to remove
contaminating red cells and additional debris. The lymphocytes were
again collected from the interface, washed twice, and saved for the
isolation of total cellular RNA.
Histopathology
Spinal cords from unimmunized B10.PL mice or from B10.PL mice treated with 1,25-(OH)2D3 or controls were removed and fixed in 10% formalin. Paraffin-embedded tissue sections were prepared and stained in luxol fast blue and periodic acid Shiffs reagent by the University of Wisconsin Veterinary Science Department. Stained sections were viewed by two individuals who were blinded as to the source of the spinal cords. Inflammation was scored as follows: 0, no sign of inflammation; 1, mild inflammation; 2, discrete lesions with substantial inflammation; and 3, multiple lesions with extensive inflammation.
Cytokine ELISAs
ELISAs were used to measure IFN-, TNF-, and TGF-ß1 (24).
The IFN- ELISA was performed exactly as described (25). The capture
and detection Abs for the TNF- ELISA were purchased from Genzyme
(Cambridge, MA). Color development was with
avidin-ß-D-galactosidase and
p-nitrophenyl-ß-D-galactosidase. Murine
TGF-ß1 was detected using the human TGF-ß1 kit exactly as described
by Promega (Madison, WI). The ELISA detection limits were 1 ng/ml
IFN-, 50 pg/ml TNF-, and 500 pg/ml TGF-ß1.
Transcript analysis by quantitative competitive PCR (QC-PCR)
Cells for mRNA analysis were dissolved in acid guanidinium
thiocyanate, and total RNA was isolated by the phenol chloroform
extraction method (26). Total cellular RNA was reverse transcribed
using oligo(dT) primers, according to the manufacturer’s protocols
(Promega) and quantitated by competitive PCR. Primers and mimic DNA
specific for glyceraldehyde-3-phosphate dehydrogenase (G3PDH), IL-4,
IFN-, TNF-, and TGF-ß1 were obtained from Clontech Laboratories
(Palo Alto, CA) (27, 28). Competitive cDNA mimics, which included the
G3PDH, IL-4, TNF-, IFN-, and TGF-ß1 primer sequences adjoining
a neutral DNA segment, were serially diluted and added to test cDNA
aliquots (26, 27). The authentic product to mimic bp sizes were 983/600
for G3PDH, 306/544 for IL-4, 333/500 for IFN-, 354/500 for TNF-,
and 525/390 for TGF-ß1. The mixture was amplified under predetermined
optimal conditions and the products were resolved by 1.5% agarose gel
electrophoresis and ethidium bromide stained. The cytokine bands were
identified by size with respect to m.w. standards. The mimic DNA
dilution that yielded a band with a fluorescence intensity that matched
the cytokine band was used to calculate cytokine cDNA copy number. The
G3PDH transcript quantitation served as a control for reverse
transcription efficiency. Values are reported as cytokine cDNA copies
per 1000 copies of G3PDH cDNA.
Statistics
Where possible, values reported were averages from multiple mice
or experiments. Because of the variability in EAE induction, peak
severity, and cytokine gene expression from one experiment to another,
some values were reported as the values from one representative
experiment of two to seven experiments. Again where possible
statistical analyses were done using a statistics program for the
Macintosh computer (STATVIEW STUDENT). The unpaired two group
Student’s t test (and confirmed using the Mann-Whitney
U test) was done and values of 
< 0.05 were considered
statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When the 1,25-(OH)2D3 treatment was
started at the same time as EAE induction, the spinal cord sections
looked identical to unimmunized B10.PL spinal cords (data not shown).
The absence of inflammatory infiltrates from spinal cord sections of
1,25-(OH)2D3-treated mice reflected the absence
of EAE symptoms (7). When 1,25-(OH)2D3 was
started at the first symptoms of EAE, the histology scores from control
mice were significantly ( = 0.0001) higher than those from
1,25-(OH)2D3-treated mice (Table I
). The differences between the control-
and 1,25-(OH)2D3-treated animal histology
scores at day 14 posttreatment were less striking and statistically
insignificant (Table I). In all cases, the histopathology scores
reflected the visual EAE severity scores in our mice (Table I).
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At various times postimmunization, the LNs draining the site of
injection from four to eight mice were collected, pooled, and counted.
The number of cells recoverable in the LNs of control mice started
between 1 and 2 x 107 cells/mouse and doubled by
10 to 14 days after immunization (Fig. 1A). Sampling on days
14 and 21 was repeated three and four times, respectively. LNs of
control mice yielded 2.6 ± 1.3 (107) cells/mouse on
day 14 and 3.9 ± 0.2 (107) cells/mouse on day 21
postimmunization. The number of cells recoverable from
1,25-(OH)2D3-fed mice did not increase with
time postimmunization (Fig. 1A). LNs of
1,25-(OH)2D3-fed mice yielded 1.1 ± 0.5
(107) cells/mouse on day 14 and 1.8 ± 0.7
(107) cells/mouse on day 21 postimmunization. The control
cell yields were significantly ( = 0.008) higher than
1,25-(OH)2D3 cell yields by day 21
postimmunization. 1,25-(OH)2D3 treatment
inhibited the in vivo expansion of LN cells both when
1,25-(OH)2D3 treatment was started at the time
of immunization (Fig. 1A) and 1 wk after immunization
(Fig. 1B). Although the total number of cells
recoverable from the LN of 1,25-(OH)2D3-treated
mice was fewer than from controls, the proportion of CD4+,
CD8+, Thy-1+7, and class II+ cells were not
different in the control- and
1,25-(OH)2D3-treated animals (data not
shown).
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and TNF-
IFN- and TNF- secretion was quantitated by assaying the
amount of protein secreted in response to MBP in vitro. IFN-
secretion was first detected 7 days postimmunization and peaked 10 days
postimmunization (Fig. 2A). IFN- production
from the LN cells of 1,25-(OH)2D3-treated mice
was the same or in one case higher than IFN- secretion from the LN
cells of control mice (Fig. 2B). The magnitude of
IFN- secreted varied greatly from experiment to experiment but the
amount of IFN- secreted in cells from
1,25-(OH)2D3-treated mice was in all but one
case (Fig. 2B, 15 of 16 experiments) identical to that of
controls. The number of IFN- transcripts was the same or lower
(statistically not different) in CNS samples from
1,25-(OH)2D3-treated mice when compared with
CNS samples from controls (Table II).
Overall, the IFN- response of the LN cells reflected the IFN-
response of cells from the CNS.
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secretion was first detected
after 14 days of immunization, and TNF- decreased thereafter (data
not shown). On day 14 postimmunization, cells from control mice
secreted 242 ± 15 pg/ml of TNF- while cells from
1,25-(OH)2D3-treated mice secreted 194 ±
59 pg/ml of TNF-. Although the differences were small, the amount of
TNF- secretion by LN cells from control mice was always higher (10
of 10 experiments) than that from
1,25-(OH)2D3-treated mice (data not shown). In
the CNS, the number of TNF- transcripts detectable from
1,25-(OH)2D3- and control-treated mice was low
and not significantly different (Table II
). TGF-ß1 and IL-4
Transcripts for IL-4 were first detected in the LN 21 days after
immunization. Two weeks of 1,25-(OH)2D3
treatment increased IL-4 transcripts 25-fold over the controls (Fig. 3). Although the overall magnitude of the
IL-4 response varied from experiment to experiment, this was a highly
reproducible result. In six of six experiments, long-term
1,25-(OH)2D3-treatment increased IL-4
transcripts 3–25-fold higher then controls. IL-4 transcripts were
detectable in samples from 1,25-(OH)2D3-treated
but not control CNS samples. This was a significant difference (Table II, = 0.0002).
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). Eightfold more
TGF-ß1 transcripts were detected in the LNs of
1,25-(OH)2D3-treated mice than controls 21 days
after immunization (Fig. 4). Although the overall magnitude of the
response varied from experiment to experiment, this was a highly
reproducible result. In seven of seven experiments, TGF-ß1
transcripts were 4- to 24-fold higher in LN of mice treated long-term
with 1,25-(OH)2D3 compared with controls.
Similarly, TGF-ß1 transcripts were 5- to 6-fold higher (significant
= 0.05) in CNS samples from
1,25-(OH)2D3-treated mice than in controls
(Table II). Because TGF-ß1 is extensively posttranscriptionally
regulated, we attempted to confirm our transcript measurements by
quantitating TGF-ß1 protein. MBP-specific TGF-ß1 protein was
undetectable from LN samples of
1,25-(OH)2D3-fed and control-fed mice at any
time postimmunization (data not shown). LPS stimulation of resident
peritoneal cells from 1,25-(OH)2D3-treated mice
did make detectable TGF-ß1 protein (630 ± 70 pg/ml), while
peritoneal cells from control-treated mice did not (
500 pg/ml).
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1,25-(OH)2D3 treatment of mice with
severe EAE caused a rapid decline in their symptoms (Fig. 5) compared with controls. Three of 10
controls but only 1 of 10 1,25-(OH)2D3-treated
mice developed severe EAE (scored 5) and were sacrificed within 3 to 5
days of treatment. By day 6
post-1,25-(OH)2D3-treatment, the remaining 7
controls had more severe EAE ( 0.05) than the 9 remaining
1,25-(OH)2D3-treated mice. The 7 control mice
underwent a short-lived recovery at day 11 posttreatment (significant
= 0.01, then day 10 EAE scores), followed by a relapse. The
1,25-(OH)2D3-treated mice maintained a low
level of symptoms as long as the diet was continuously supplemented
with 1,25-(OH)2D3. Twenty-four hours after
1,25-(OH)2D3 treatment, the LN and serum were
collected from 5 individual mice per treatment group. Serum calcium was
raised slightly in the 1,25-(OH)2D3-treated
mice, confirming the effectiveness of the treatment (Table III). IFN- and TNF- transcripts
were not significantly different following
1,25-(OH)2D3 treatment (Table III). IL-4
transcripts were fourfold higher (although statistically not different)
in the 1,25-(OH)2D3-treated mice compared with
controls. TGF-ß1 transcripts were fivefold higher and significantly
( 0.04) higher in mice receiving a single injection of
1,25-(OH)2D3 24 h before sacrifice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or TNF- made on a per cell
basis at any stage of EAE disease. However, control mice had as much as
twice the number of mononuclear cells in the LNs than
1,25-(OH)2D3-treated mice, and the net effect
for control mice would be a Th1 response that is twice that of the
1,25-(OH)2D3-treated mice. We conclude that
1,25-(OH)2D3 is not negatively regulating the
Th1 cells that drive the encephalitogenic process, although this
hormone may down-regulate the Th1 response through a modest effect on
cellular numbers.
TGF-ß1 is a potent modulator of cell growth (29). This cytokine
inhibits the proliferation and differentiation of T and B cells (29),
and antagonizes the effects of specific inflammatory cytokines,
including TNF-, IFN-, IL-1, IL-2, and IL-6 (30). More
specifically, TGF-ß1 is a critical regulator of autoimmune disease
(30). Exogenous TGF-ß1 can delay the onset and decrease the severity
of EAE and arthritis in mice (16, 30, 31). Conversely, neutralization
of TGF-ß1 in vivo increases the severity of these two autoimmune
diseases in the mouse (17, 30). In the present investigation,
supplementation with 1,25-(OH)2D3 increased the
number of transcripts of TGF-ß1 mRNA in mononuclear cells both from
sensitized LN and from affected central nervous tissue. This phenomenon
was apparent in LN cells regardless of the state of disease at the time
the hormone was first administered. Moreover,
1,25-(OH)2D3 was effective within only 24
h, an outcome that suggests a direct influence on cytokine production.
Our current hypothesis, therefore, is that the actions of
1,25-(OH)2D3 in preventing and treating EAE may
be in part a result of positive regulation of TGF-ß1.
We did not detect an MBP-specific increase in TGF-ß1 protein in vitro, but LPS stimulated TGF-ß1 protein secretion in peritoneal exudate cells of mice given 1,25-(OH)2D3 supplements. It therefore seems likely that in our experiments TGF-ß1 is coming from a macrophage and not from a T cell. LN cells are greater then 90% lymphocytes and have few macrophage cells relative to peritoneal cells. This is important in view of the finding that macrophage cells constitutively produce vitamin D receptor, while among T cells, activation is required for expression of the vitamin D receptor (32, 33). It seems likely that in EAE, 1,25-(OH)2D3 may be up-regulating the production of TGF-ß1 by macrophage cells.
Long-term in vivo 1,25-(OH)2D3 treatment
increased IL-4 transcripts in the LN and CNS compared with controls.
Our data suggest that the effect of
1,25-(OH)2D3 on IL-4 may be indirect. The
increase in IL-4 24 h after 1,25-(OH)2D3
injection was not significantly different from controls (Table III). We
hypothesize that 1,25-(OH)2D3-driven TGF-ß1
production by macrophage cells might make the T cell microenvironment
more conducive to Th2-type cell differentiation. Experiments are
underway to address this point.
We have previously shown that 1,25-(OH)2D3 can
prevent and diminish the severity of EAE (7). The present results
suggest that 1,25-(OH)2D3 acts by positively
regulating the anti-encephalitogenic cytokines IL-4 and TGF-ß1.
We were unable to obtain results consistent with a direct effect of
1,25-(OH)2D3 on the encephalitogenic Th1
effector cell in terms of either IFN- or TNF- expression. As a
result of our 1,25-(OH)2D3 treatments, the
numbers of cells recoverable from the LN and the number of inflammatory
lesions discernible in histopathology sections were dramatically
reduced. This could be due, in part, to increased production of
TGF-ß1, a cytokine known to inhibit T and B cell proliferation (16, 17). Based on the ability of TGF-ß1 to ameliorate EAE (16), we
hypothesize that 1,25-(OH)2D3-mediated
up-regulation of TGF-ß1 production might be the immunobiologic
mechanism underlying the efficacy of
1,25-(OH)2D3 for halting the progression
of EAE.
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Acknowledgments |
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Footnotes |
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2 Present address: Dr. William D. Woodward, Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada NIG 2W1.
3 Address correspondence and reprint requests to Dr. Hector F. DeLuca, Department of Biochemistry, University of Wisconsin-Madison, 420 Henry Mall, Madison, WI 53706. E-mail address:
4 Abbreviations used in this paper: MS, multiple sclerosis; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; LN, lymph node; G3PDH, glyceraldehyde-3-phosphate dehydrogenase.
Received for publication August 8, 1997. Accepted for publication February 3, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expression by resident microglia and infiltrating leukocytes in the central nervous system of mice with experimental allergic encephalomyelitis: regulation by Th1 cytokines. J. Immunol. 154:944.[Abstract]
plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis. J. Immunol. 157:3223.[Abstract]
gene are susceptible to the induction of experimental autoimmune encephalomyelitis (EAE). J. Immunol. 156:5.[Abstract]
and the proliferation of human peripheral blood leukocytes while enhancing the surface expression of HLA class II molecules. J. Endocrinol. Invest. 12:329.[Medline]
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M. Cippitelli, C. Fionda, D. Di Bona, F. Di Rosa, A. Lupo, M. Piccoli, L. Frati, and A. Santoni Negative Regulation of CD95 Ligand Gene Expression by Vitamin D3 in T Lymphocytes J. Immunol., February 1, 2002; 168(3): 1154 - 1166. [Abstract] [Full Text] [PDF]
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T. P. Staeva-Vieira and L. P. Freedman 1,25-Dihydroxyvitamin D3 Inhibits IFN-{gamma} and IL-4 Levels During In Vitro Polarization of Primary Murine CD4+ T Cells J. Immunol., February 1, 2002; 168(3): 1181 - 1189. [Abstract] [Full Text] [PDF]
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 H. F. DELUCA and M. T. CANTORNA Vitamin D: its role and uses in immunology FASEB J, December 1, 2001; 15(14): 2579 - 2585. [Abstract] [Full Text] [PDF]
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A. Boonstra, F. J. Barrat, C. Crain, V. L. Heath, H. F. J. Savelkoul, and A. O'Garra 1{alpha},25-Dihydroxyvitamin D3 Has a Direct Effect on Naive CD4+ T Cells to Enhance the Development of Th2 Cells J. Immunol., November 1, 2001; 167(9): 4974 - 4980. [Abstract] [Full Text] [PDF]
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 M. T. Cantorna, C. Munsick, C. Bemiss, and B. D. Mahon 1,25-Dihydroxycholecalciferol Prevents and Ameliorates Symptoms of Experimental Murine Inflammatory Bowel Disease J. Nutr., November 1, 2000; 130(11): 2648 - 2652. [Abstract] [Full Text] [PDF]
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M. T. Cantorna Vitamin D and Autoimmunity: Is Vitamin D Status an Environmental Factor Affecting Autoimmune Disease Prevalence? Experimental Biology and Medicine, March 1, 2000; 223(3): 230 - 233. [Abstract] [Full Text]
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M. T. Cantorna, J. Humpal-Winter, and H. F. DeLuca Dietary Calcium Is a Major Factor in 1,25-Dihydroxycholecalciferol Suppression of Experimental Autoimmune Encephalomyelitis in Mice J. Nutr., November 1, 1999; 129(11): 1966 - 1971. [Abstract] [Full Text]
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 G. JONES, S. A. STRUGNELL, and H. F. DeLUCA Current Understanding of the Molecular Actions of Vitamin D Physiol Rev, October 1, 1998; 78(4): 1193 - 1231. [Abstract] [Full Text] [PDF]
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T. F. Meehan and H. F. DeLuca CD8+ T cells are not necessary for 1alpha ,25-dihydroxyvitamin D3 to suppress experimental autoimmune encephalomyelitis in mice PNAS, April 16, 2002; 99(8): 5557 - 5560. [Abstract] [Full Text] [PDF]
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