Dumitru Branisteanu

The vitamin D receptor gene FokI polymorphism: Functional impact on the immune system

1α,25‐Dihydroxyvitamin D3 (1,25(OH)2D3) has important effects on the growth and function of multiple cell types. These pleiotropic effects of 1,25(OH)2D3 are mediated through binding to the vitamin D receptor (VDR). Several polymorphisms of the human VDR gene have been identified, with the FokI polymorphism resulting in VDR proteins with different structures, a long f‐VDR or a shorter F‐VDR. The aim of this study was to investigate the functional consequences of the FokI polymorphism in immune cells. In transfection experiments, the presence of the shorter F‐VDR resulted in higher NF‐κB‐ and NFAT‐driven transcription as well as higher IL‐12p40 promoter‐driven transcription. Marginal differences were observed for AP‐1‐driven transcription, and no differential effects were observed for transactivation of a classical vitamin D‐responsive element. Concordantly, in human monocytes and dendritic cells with a homozygous short FF VDR genotype, expression of IL‐12 (mRNA and protein) was higher than in cells with a long ff VDR genotype. Additionally, lymphocytes with a short FF VDR genotype proliferated more strongly in response to phytohemagglutinin. Together, these data provide the first evidence that the VDR FokI polymorphism affects immune cell behavior, with a more active immune system for the short F‐VDR, thus possibly playing a role in immune‐mediated diseases.

Analogs of 1,25-dihydroxyvitamin D3 as dose-reducing agents for classical immunosuppressants.

BACKGROUND Most immunosuppressants have a narrow margin between efficacy and side effects. A major goal in the development of immunomodulatory strategies is the discovery of combinations of drugs exerting synergistic immunomodulatory effects. The active form of vitamin D, 1,25(OH)2D3, is an immunomodulator that interacts with T cells but mainly targets antigen-presenting cells. We have demonstrated synergism between 1,25(OH)2D3 and cyclosporine, rapamycin, and FK506. The aim of this study was to investigate whether this synergism could be observed with other immunosuppressants (mycophenolate mofetil, leflunomide, and the methylxanthine A802715) and whether analogs of 1,25(OH)2D3 share this synergistic capacity in vivo. METHODS In vitro, the median effect analysis was applied to the inhibition of phytohemagglutinin A-induced lymphocyte proliferation. In vivo, synergism between analogs of 1,25(OH)2D3 and cyclosporine or mycophenolate mofetil was evaluated in experimental autoimmune encephalomyelitis. RESULTS In vitro, all combinations with 1,25(OH)2D3 were synergistic. The strongest synergism was seen with the inhibitors of interleukin 2 secretion, cyclosporine and FK506 (indexes 0.16 and 0.27, respectively). The weakest synergism was observed in combinations using A802715, a second-signal inhibitor (index 0.52), or the nucleotide synthesis inhibitor mycophenolate mofetil (index 0.43). In vivo, analogs of 1,25(OH)2D3 share the in vitro-observed synergism with 1,25(OH)2D3. Moreover, the differences in synergism with different immunomodulators were also present in vivo, where the best synergism was again seen in combination with cyclosporine (up to 100% paralysis protection). CONCLUSIONS These data confirm that 1,25(OH)2D3 and its analogs are potent dose-reducing drugs for other immunomodulators, making them potentially interesting for clinical use in autoimmunity and transplantation.

Prevention of murine experimental allergic encephalomyelitis: cooperative effects of cyclosporine and 1 α, 25-(OH)2D3

The hormone 1 alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) has immune modulatory activities in vitro and in vivo, and can prevent or delay the onset of experimental or spontaneous autoimmune diseases. At therapeutical doses, however, hypercalcemic side effects are found. The present experiments examined the effects of combined treatment with subtherapeutic doses of cyclosporine A (CsA) and 1,25(OH)2D3 on the evolution of experimental autoimmune encephalomyelitis (EAE) in SJL mice. 1,25(OH)2D3 at 5 micrograms/kg body weight (given by i.p. injection every 2 days) prevented the appearance of paralysis in 70% of the treated mice. The treatment with 1,25(OH)2D3 at 2 micrograms/kg/2 days alone had less substantial protective effects (22% disease-free animals versus 5% in the control group). However, when this subtherapeutic dose was associated to treatment with a daily dose of CsA (2 or 5 mg/kg/day), which by itself was subtherapeutic (24 and 50% disease-free animals, respectively), the association of both drugs led to near-total protection (86% disease-free animals when combined with the highest dose of CsA). When an alternate day administration schedule (CsA at 10 mg/kg and 1,25(OH)2D3 at 2 micrograms/kg, each given on alternate days from day -3 to +19 after disease induction) was used, all treated mice were completely protected clinically and histologically. The two drugs also showed additive effects on serum osteocalcin and urinary calcium and desoxypyridinoline excretion, but not on serum calcium concentration. Our experiments demonstrate that 1,25(OH)2D3 might be a potential dose-reducing agent for CsA in immunosuppressive therapy.

Partial prevention of active Heymann nephritis by lα, 25 dihydroxyvitamin D3

The hormone lα, 25 dihydroxyvitamin D3 (1,25(OH)2DO3 has potent immunosuppressivc effects in vitro. Recent publications also described a protective effect of the hormone in various animal models of immune‐mediated diseases. To test its in vitro activity we induced active Heymann nephritis in Lewis rats that were either untreated or treated with 1,25(OH)2D3 or its synthetic 20‐epi analogue, KH1060. Treatment with cyclosporine A (CsA) was used as an immunosuppressive control. In this nephrotic model the administration of 1,25(OH)2D3 (0.5 μg/kg body weight) given on alternate days during the first 13 days after active immunization significantly reduced the proteinuria as measured by weeks 7–9. This reduction was comparable to the reduction observed in rats treated with CsA (20 mg/kg) on alternate days. A second series of experiments with 1,25(OH)2D3 confirmed these findings. The level of autoantibodies was found to be significantly suppressed during the treatment time in the CsA (20 mg/kg) group, whereas the limit of significance (P=0.06) was reached in the 1,25(OH)2D3 groups that developed less proteinuria. The administration of 1,25(OH)2D3 transiently increased the mean serum calcium Concentration with 2.5 mg/dl above the pretreatmcnt values, and the urinary calcium excretion by a factor of 3–5 during the short treatment time. Treatment with the analogue KH 1060 did not reduce the proteinuria significantly. Our experiments add evidence to the hypothesis that 1,25(OH)2D3 in pharmacological doses has immunosupprcssive potency.

Novel insights in the immune function of the vitamin D system: Synergism with interferon-beta

The 1,25(OH)(2)D(3) analog, TX527 (19-nor-14,20-bisepi-23-yne-1,25(OH)(2)D(3)), has an interesting dissociation profile between its potent immunomodulatory and its calcemic effects in vivo. The strong immunomodulatory potency of TX527 is reflected by its ability to attenuate experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). At present most MS patients are being treated with systemic IFN-beta administration. The aim of this study was to investigate whether combining IFN-beta with TX527 could empower its EAE-protective effects. We evaluated also combinations with the standard immunosuppressant cyclosporin A (CsA). EAE was induced in SJL mice by PLP immunization, treatment was started 3 days before disease induction. The TX527+IFN-beta combination resulted in significant disease protection which was superior to the effect of both treatment separately. No disease amelioration, even aggravation, was obtained with the IFN-beta+CsA combination. By adding TX527 to the IFN-beta+CsA combination near complete protection from EAE was achieved (100% protection from paralysis, mean maximal score of 1.8+/-1.5, both p<0.05 versus controls and all individual treatments). From these data we conclude that adding TX527 to an IFN-beta and/or CsA treatment results in clear additional immunomodulatory effects in EAE prevention and is therefore a potentially interesting candidate to be considered in clinical intervention trials in MS.

Combination of a 1,25-dihydroxyvitamin D3 analog and a bisphosphonate prevents experimental autoimmune encephalomyelitis and preserves bone

The vitamin D analog TX527 (19-nor-14,20-bis epi-23-yne-1,25(OH)(2)D(3)), decreased disease severity (P < 0.001) and postponed disease onset (P < 0.0001) in SJL mice in which experimental autoimmune encephalomyelitis was induced. Levels of IFN-gamma and IL-2 mRNA were decreased in spinal cord and spleen in the analog-treated mice, suggesting a Th(1)-targeted effect. Adding the bisphosphonate pamidronate did not affect analog-protective efficacy, but completely prevented TX527-caused acceleration of bone turnover and increased total bone mineral content as well as femoral mineral and calcium content (P < 0.01). Less calcemic analogs of 1,25-dihydroxyvitamin D(3), in combination with bone sparing products such as bisphosphonates allow immune modulation in vivo without affecting bone.

Synergism between sirolimus and 1,25-dihydroxyvitamin D3 in vitro and in vivo

The active form of vitamin D, 1 alpha, 25-(OH)2D3, displays immunomodulatory effects in vitro and in vivo at pharmacological levels. We evaluated the dose-effect relationship of 1,25(OH)2D3 and sirolimus (rapamycin, RAP) in vitro, on the inhibition of PHA-stimulated PBMC proliferation, by using the median effect analysis. Pharmacological concentrations of 1,25(OH)2D3 (between 10(-9) and 3 x 10(-6) M) interacted synergistically with RAP (combination index value of 0.01 for 50% suppression of PBMC proliferation). In vivo, the effect of 1,25(OH)2D3 and RAP combinations on the evolution of experimental allergic encephalomyelitis in SJL mice was analyzed. 1,25(OH)2D3, given i.p., in monotherapy, at a dose of 2 micrograms/kg every two days, from day -3 until day +19 after disease induction, or RAP, injected daily at a dose of 0.3 mg/kg for the same period, decreased EAE incidence (paralysis in 70 and 55% of the animals, respectively, versus 98% in the placebo treated group, p < 0.001). The combination treatment using the two drugs in these subtherapeutical doses provided near-total clinical (8% paralysis, p < 0.001 compared to monotherapy with 1,25(OH)2D3 or RAP) and histological protection, comparable to that obtained with RAP in monotherapy at a threefold higher dose (1 mg/kg/d). When the two drugs were given using an alternate day administration schedule (RAP at 0.6 mg/kg and 1,25(OH)2D3 at 2 micrograms/kg, each given on alternate days from day -3 to 19), near total protection was again obtained (13% paralysis, p < 0.001 versus control). These in vitro and in vivo data support the existence of synergistic interactions between 1,25(OH)2D3 and RAP. Considering the narrow therapeutic windows of both RAP and vitamin D-related compounds in autoimmune disease models, combinations of these drugs could find clinical application in reducing their individual therapeutically efficient doses to non-toxic levels.

Combination of Vitamin D Analogues and Immunosuppressants

The active form of vitamin D, calcitriol (1,25-dihydroxyvitamin D3), has important immunomodulating effects in vitro and in animal models of transplantation and autoimmune diseases. There is increasing evidence of cooperative effects between calcitriol and other immunosuppressants. New synthetic vitamin D analogues show much attenuated toxicity when compared with calcitriol while retaining their immunosuppressive properties. Subtherapeutic doses of such analogues might therefore be used as dose-reducing agents for classical immunosuppressants.

Hen Egg White Lysozyme Vaccination Induces Acute Shock in NOD Mice

Abstract: To investigate whether vaccination could induce lethal shock and which mechanisms are involved in this phenomenon, we tested a panel of autoantigens or diabetes‐irrelevant peptides or proteins in nonobese diabetic (NOD), Balb/c, and C57Bl/6 mice. Of the antigens tested, only nondiabetogenic hen egg white lysozyme induced a severe form of shock exclusively in NOD mice. The mechanism involved is suggestive of a Th2‐mediated anaphylactic reaction possibly connected to activation of PAF and triggering of DIC.