Anti-Thymocyte Globulin Treatment Augments 1,25-Dihydroxyvitamin D3 Serum Levels in Patients Undergoing Hematopoietic Stem Cell Transplantation

Application of anti-thymocyte globulin (ATG) is a widely used strategy for the prevention of graft-versus-host disease (GvHD). As vitamin D3 serum levels are also discussed to affect hematopoietic stem cell transplantation (HSCT) outcome and GvHD development, we analysed a possible interplay between ATG treatment and serum levels of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 in 4 HSCT cohorts with different vitamin D3 supplementation. ATG is significantly associated with higher serum level of 1,25-dihydroxyvitamin D3 around HSCT (day -2 to 7, peri-transplant), however only in patients with adequate levels of its precursor 25-hydroxyvitamin D3. ATG exposure had no impact on overall survival in patients supplemented with high dose vitamin D3, but was associated with higher risk of one-year treatment-related mortality (log rank test p=0.041) in patients with no/low vitamin D3 supplementation. However, the difference failed to reach significance applying a Cox-model regression without and with adjustment for baseline risk factors (unadjusted P=0,058, adjusted p=0,139). To shed some light on underlying mechanisms, we investigated the impact of ATG on 1,25-Dihydroxyvitamin D3 production by human dendritic cells (DCs) in vitro. ATG increased gene expression of CYP27B1, the enzyme responsible for the conversion of 25-hydroxyvitamin D3 into 1,25-dihydroxyvitamin D3, which was accompanied by higher 1,25-dihydroxyvitamin D3 levels in ATG-treated DC culture supernatants. Our data demonstrate a cooperative effect of 25-hydroxyvitamin D3 and ATG in the regulation of 1,25-dihydroxyvitamin D3 production. This finding may be of importance in the context of HSCT, where early high levels of 1,25-dihydroxyvitamin D3 levels have been shown to be predictive for lower transplant related mortality and suggest that vitamin D3 supplementation may especially be important in patients receiving ATG for GvHD prophylaxis.

1,25-dihydroxyvitamin D3 Ameliorates Lupus Nephritis Through Inhibiting NF-κB and MAPKs Signaling Pathways in MRL/lpr Mice

Background: Lupus nephritis (LN) is a common and serious complication of systemic lupus erythematosus (SLE). However, the etiology and pathogenesis of LN remain unknown. 1,25-dihydroxyvitamin D3（1,25-(OH)2-VitD3）is the active form of vitamin D, which has been known to have important functions in inflammation and immune diseases. In this study, we investigated Its protective effects and underlying mechanism in MRL/lpr mice, a well-studied animal model for lupus.Methods: At the age of 11 weeks, forty-eight MRL/lpr mice were randomly divided into two groups with 24 mice per group: the VitD3-treated group and control group. Mice in the VitD3-treated group received 4μg/kg 1,25-dihydroxyvitamin D3 in 1％ dimethyl sulfoxide (DMSO) intraperitoneal injection twice a week for 3 weeks (mice were executed at 0,2,4,6 weeks after treatment); mice in the control group treated with intraperitoneal injection of 1% DMSO for 3 weeks (mice were executed at 0,2,4,6 weeks after injections). The mice were sacrificed and the serum and kidney samples were collected respectively at planned intervals. Then the skin lesions, histological changes, inflammatory factors (TNF-α, IL-17) and immunological markers (A-ds DNA, C3, IgG, IgM) with time were analyzed between the groups. Furthermore, the NF-κB and MAPKs signaling pathways were also detected to explicate the underlying mechanism. Results: Compared to the control group, mice in the VitD3-treated group showed less skin lesions, less kidney injury, lower serum anti-ds DNA antibody, lower inflammatory cytokines TNF-α, IL-17 and higher serum complement C3; they also had less deposition of IgG, IgM and C3 within glomeruli. Moreover, the expressions of NF-κB and MAPKs signaling pathways were decreased, while those levels were increased with time.Conclusion: This study shows that 1,25-dihydroxyvitamin D3 exerts a protective effect against lupus nephritis via regulating the NF-κB and MAPKs signaling pathways, which will be developed as a potential agent for the treatment of Lupus nephritis. And the relationship between lupus activity and NF-kB and MAPKs signaling pathways with time was revealed.

1,25-Dihydroxyvitamin D3 and 20-Hydroxyvitamin D3 Upregulate LAIR-1 and Attenuate Collagen Induced Arthritis

Vitamin D plays a crucial role in regulation of the immune response. However, treatment of autoimmune diseases with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] doses sufficient to be effective is prohibitive due to its calcemic and toxic effects. We use the collagen-induced arthritis (CIA) model to analyze the efficacy of the noncalcemic analog of vitamin D, 20S-hydroxyvitamin D3 [20S(OH)D3], as well as 1,25(OH)2D3, to attenuate arthritis and explore a potential mechanism of action. Mice fed a diet deficient in vitamin D developed a more severe arthritis characterized by enhanced secretion of T cell inflammatory cytokines, compared to mice fed a normal diet. The T cell inflammatory cytokines were effectively suppressed, however, by culture of the cells with 20S(OH)D3. Interestingly, one of the consequences of culture with 1,25(OH)2D3 or 20S(OH)D3, was upregulation of the natural inhibitory receptor leukocyte associated immunoglobulin-like receptor-1 (LAIR-1 or CD305). Polyclonal antibodies which activate LAIR-1 were also capable of attenuating arthritis. Moreover, oral therapy with active forms of vitamin D suppressed arthritis in LAIR-1 sufficient DR1 mice, but were ineffective in LAIR-1−/− deficient mice. Taken together, these data show that the effect of vitamin D on inflammation is at least, in part, mediated by LAIR-1 and that non-calcemic 20S(OH)D3 may be a promising therapeutic agent for the treatment of autoimmune diseases such as Rheumatoid Arthritis.

Effect of 1,25-Dihydroxyvitamin D3 on Stem Cells from Human Apical Papilla: Adhesion, Spreading, Proliferation, and Osteogenic Differentiation

Currently, it still remains a difficult problem to treat apical insufficiency of young permanent teeth resulted from pulp necrosis or periapical periodontitis. Previous studies have demonstrated that the treatment of revascularization using stem cells from apical papilla (SCAPs) results in increased root length and thickness of traumatized immature teeth and necrotic pulp. In this study, we investigated the role of 1,25-dihydroxyvitamin D3 in regulating the adhesion, spreading, proliferation, and osteogenic differentiation of SCAP, laying the foundation for subsequent clinical drug development. The immature tooth samples were collected in clinical treatment. SCAPs with stable passage ability were isolated and cultured. The multidifferentiation potential was determined by directed induction culture, while the stem cell characteristics were identified by flow cytometry. There were three groups: group A—SCAPs general culture group; group B—SCAPs osteogenesis induction culture group; and group C—SCAPs osteogenesis induction culture+1,25-dihydroxyvitamin D3 group, and the groups were compared statistically. The proliferation of SCAPs in each groups was detected through CCK-8 assay. RT-qPCR was used to detect the transcription levels of Runx2, ALP, Col I, and OCN of SCAPs in each groups. Results exhibited that the isolated SCAPs had multidifferentiation potential and stem cell characteristics. After 24 h culturing, cells in group C spread better than those in groups A and B. The proliferation activity of SCAPs factored by CCK-8 ranked as group C > group B > group A , while the transcription levels of Runx2, ALP, Col I, and OCN leveled as group C > group B > group A . These results suggested that 1,25-dihydroxyvitamin D3 can significantly promote the adhesion, spreading, and proliferation of SACPs and improve the osteogenic differentiation of SCAPs by means of regulating upward the transcription level of osteogenic differentiation marker.

Anti-Tumor Effects of Ginsenoside 20(S)-Protopanaxadiol and 1,25-Dihydroxyvitamin D3 Combination in Castration Resistant Prostate Cancer

In spite of possessing desirable anticancer properties, currently, limited clinical success has been achieved with 20(S)-protopanaxadiol (aPPD) and 1,25-dihydroxyvitamin D3 (calcitriol). This study is designed to evaluate if the combination of aPPD with calcitriol can inhibit human prostate cancer xenograft growth by using nuclear receptor signaling. Athymic male nude mice were utilized to establish an androgen-independent human prostate cancer C4-2 cell castration-resistant prostate cancer (CRPC) xenograft model. Mice were treated orally for six weeks with 70 mg/kg aPPD administered once daily or three times per week with 4 µg/kg calcitriol or in combination or only with vehicle control. Contrary to our expectations, calcitriol treatment alone increased C4-2 tumor growth. However, the addition of calcitriol substantially increased aPPD-mediated tumor growth suppression (76% vs. 53%, combination vs. aPPD alone). The combination treatment significantly increased levels of cleaved caspase-3 apoptotic marker compared to vehicle-treated or aPPD-treated C4-2 tumors. The mechanistic elucidations indicate that tumor inhibition by the aPPD and calcitriol combination was accompanied by elevated vitamin D receptor (VDR) protein expression. In silico data suggest that aPPD weakly binds to the native LBD pocket of VDR. Interestingly, the combination of aPPD and calcitriol activated VDR at a significantly higher level than calcitriol alone and this indicates that aPPD may be an allosteric activator of VDR. Overall, aPPD and calcitriol combination significantly inhibited tumor growth in vivo with no acute or chronic toxic effects in the C4-2 xenograft CRPC nude mice. The involvement of VDR and downstream apoptotic pathways are potential mechanistic routes of antitumor effects of this combination.