Intramammary treatment with 1,25-dihydroxyvitamin D3 increases the expression of host defense genes in dairy cattle mammary immune cells (2023)

The objectives were to determine the effect of supplementation of 2 sources of vitamin D, cholecalciferol (CH) or calcidiol (CA), at a dose of 1 (1 mg) or 3 mg/day (3 mg) before administration on plasma concentrations of vitamin D metabolites, measurements of innate immune function and leukocyte mRNA expression. Mating Holstein cows (n = 99) were treated daily from 250 days gestation until calving as a supplement containing 1 or 3 mg CH (CH1 or CH3) or CA (CA1 or CA3). Plasma vitamin D concentrations, blood immune cell populations, markers of cell adhesion and granulocyte phagocytosis and oxidative burst were assessed before and after delivery. Leukocyte mRNA expression was determined at day 270 of gestation and day 3 postpartum for genes involved in cell migration, pathogen recognition receptors, cell signaling, cytokines, antimicrobial mechanisms, oxidative burst, and the metabolism of Ca and vitamin D. Vitamin D concentrations₃increased in cows given CH, while those given 25-hydroxyvitamin D₃increased in cows receiving CA. The percentage of granulocytes to total leukocytes differed with the amount of vitamin D before (1 mg = 24.5 vs. 3 mg = 37.9%) and after delivery (1 mg = 22.0 vs. 3 mg = 31.0%), so mononuclear cells shift in the opposite direction before (1 mg = 75.5 vs. 3 mg = 62.1%) and after delivery (1 mg = 78.0 vs. 3 mg = 69, 0%). Granulocytes showing phagocytosis (1 mg = 69.0 vs. 3 mg = 62.9%) and intensity of prepartum phagocytosis (1 mg = 7.46 vs. 3 mg = 7.28) were generally lower in cows that 3 mg compared to 1 mg. During the prenatal period, CA increased mRNA expression of genes related to cell adhesion and migration (CD44, ICAM1, ITGAL, ITGB1, LGALS8, SELL), pathogen recognition receptor (NOD2, TLR2, TLR6), cell signaling (FOS, JUN, NFKB2). , cytokine signaling (IL1B, IL1R1, IL1RN), antimicrobial mechanisms (CTSB, LYZ), and Ca metabolism (ATP2B1, STIM1, TRPV5) compared with CH. Similarly, postpartum CA increased mRNA expression of genes related to cell adhesion and migration (CXCR2, SELL, TLN1), cell signaling (AKT2), ​​cytokines (CCL2, IL1R1, ILRN), antimicrobial mechanisms (DEFB3), oxidative burst (RAC2) and calcium metabolism (CALM3) compared to CH. Supplemental vitamin D nutrition during the last 3 weeks of pregnancy altered blood leukocyte profile and attenuated granulocyte phagocytosis during the transitional period, while prenatal CA supplementation increased mRNA expression of genes involved in immune cell function, including genes associated with pathogen recognition and antimicrobial effects by leukocytes.

Periparturient cattle face an increased risk of metabolic and infectious diseases. Factors contributing to this predisposition include oxidized polyunsaturated fatty acids, also known as oxylipids, the production of which is altered during the periparturient period and in affected livestock. Changes in the production of oxylipids derived from cytochrome P450 (CYP450) enzymes are overrepresented during periods of increased disease risk and clinical illness, such as mastitis. Many of these same CYP450 enzymes further regulate the metabolism of fat-soluble vitamins, such as A, D, and E. These vitamins are critical to maintaining immune system health, but circulating concentrations are reduced near calving. Despite this, relatively little research has been done on the role of CYP450 enzymes outside the liver. The aim of this work is to describe the relative expression of 11 CYP450 genes in bovine tissues and common in vitro models of bovine cells. Eight tissue samples were collected from 3 healthy dairy cows after euthanasia. In vitro samples included primary bovine aortic and mammary endothelial cells and immortalized bovine renal and mammary epithelial cells. Quantitative real-time PCR was performed to assess basal CYP450 enzyme transcript expression. Surprisingly, CYP450 mRNA was widely expressed in all tissue samples, with predominance in the liver. In vitro expression of CYP450 was less robust, with several cell types not expressing specific CYP450 enzymes. In general, cell culture models did not reflect tissue CYP450 expression. However, when CYP450 was organized by activity, certain cell types consistently expressed specific functional groups. These data reveal broad expression of CYP450 in selected organs of healthy dairy cows. Widespread expression helps explain previous evidence of significant changes in CYP450-mediated oxylipid production and fat-soluble vitamin metabolism in the microenvironment of organs during periods of oxidative stress or disease. As such, these data form the basis for targeted functional experiments aimed at understanding specific CYP450 activity and associated therapeutic potential during periods of increased disease risk.

The objectives were to determine the effects of diet with 25-hydroxyvitamin D supplementation₃[25(OH)D₃] on serum concentrations of vitamin D metabolites and minerals, mammary immune status and responses to intramammary bacterial infection in dairy cows. Sixty pregnant lactating Holstein cows with a cell count <200,000/ml were blocked in milk yield and milk yield for days and randomly assigned to receive a daily dietary supplement containing 1 or 3 mg of vitamin D₃(1mgD of 3mgD), of 1 of 3 mg 25(OH)D₃(1mg25D or 3mg25D) for 28 days (n = 15/treatment). Cows were kept in a free-range barn and received a total mixed ration in individual feed batches. Individual dry matter intake (DMI) and milk yield were recorded daily and milk and blood samples were taken at 0, 7, 14 and 21 days after the start of treatment. After 21 days, cows receiving 1 mgD and 3 mg25D developed an intramammary infection with Streptococcus uberis. Cows were monitored for the severity of mastitis and blood and milk samples were collected every 12 hours to measure inflammation. Cows with 1mg25D and 3mg25D had a higher serum 25(OH)D₃concentrations after 21 days compared to cows receiving 1 mgD and 3 mgD (62 ± 7, 66 ± 8, 135 ± 15 and 232 ± 26 ng/ml for 1 mgD, 3 mgD, 1 mg25D and 3mg25D, respectively). Cows with 3mg25D had higher serum Ca and P concentrations after 21 days compared to other treatments (Ca = 2.38, 2.4, 2.37 and 2.48 ± 0.02 mM, 1.87, 1, 88 and 2.10 ± 0.08 mM for 1 mgD, 3 mgD, 1 mg25D and 3mg25D, respectively). Yields of milk and milk solids, DMI, body weight and serum concentrations of 1,25-dihydroxyvitamin D and Mg did not differ between treatments. The abundance of mRNA transcripts for interleukin-1β (IL1B) and inducible nitric oxide synthase (iNOS) in somatic milk cells before exposure to S. uberis was increased in cows fed 25(OH)D₃compared to cows that received vitamin D₃. In addition, IL1B, iNOS, β-defensin 7 and β-defensin 10 in milk somatic cells increase with 25(OH)D concentrations₃elevated in serum. Cows receiving 3 mg 25 D had less severe mastitis 60 and 72 hours after exposure to S. uberis than cows receiving 1 mg D. Concentrations of bacteria, somatic cells and serum albumin in post-challenge milk did not differ between treatments; however, a treatment-day interaction was detected for lactate dehydrogenase in milk. Expression of the adhesion protein CD11b on mammary neutrophils after exposure to S. uberis was higher in cows at 3mg25D compared to cows at 1mgD. CYP24A1 and iNOS transcripts in somatic milk cells during mastitis were also higher in 3 mg25D cows compared to 1 mgD cows. Nutrition 25(OH)D₃elevated serum 25(OH)D₃more effective than supplemental vitamin D₃, resulting in increased serum mineral concentrations, increased expression of vitamin D responsive genes and altered immune responses to intramammary bacterial challenge.

Bovine tuberculosis, a re-emerging infectious disease caused by Mycobacterium bovis, can be transmitted to humans. The global prevalence of M. bovis in humans is underestimated and poses a serious public health risk in developing countries. In light of this situation, it is important to note that our understanding of the immunopathogenesis of human tuberculosis can be improved by studying this disease in a bovine model. Stimulation of the innate immune system of cattle with calcitriol (1,25(OH)2D3) leads to an increase in bactericidal molecules involved in the antimicrobial activity of macrophages. However, it is not known whether the effect of calcitriol on bovine macrophages affects intracellular bacterial replication. With these considerations in mind, this study sought to investigate the specific role of calcitriol in the control of tuberculosis in bovine macrophages, hoping to reveal information applicable to human tuberculosis. As such, M. bovis infection has been shown to induce CYP27B1 and VDR gene expression in macrophages. In addition, addition of 1,25(OH)2D3 to cultures of macrophages pre-infected with mycobacteria and/or activated with LPS induced cellular nitric oxide synthase (NOS2) expression and increased nitrite concentrations, both indicators of nitric oxide (NO) production. Using a microbicidal assay, the addition of 1,25(OH)2D3 was observed to increase macrophage phagocytosis and decrease intracellular replication of mycobacteria. Therefore, our results taken together indicate that calcitriol may help stimulate the innate immune system of cattle by increasing phagocytosis and decreasing the intracellular replication of microorganisms, such as M. bovis, in macrophages via the VDR pathway.

Vitamin D signaling in response to pathogen-associated molecules contributes to the activation of innate immune responses of bovine monocytes. We hypothesized that lipopolysaccharide (LPS) from mastitis-associated bacteria in dairy cows activates the vitamin D pathway in udder innate immune cells and increases 25-hydroxyvitamin D availability.₃[25(OH)D₃] would increase the expression of genes related to vitamin D. The aim of this experiment was to determine the effects of intramammary LPS and 25(OH)D₃treatments of vitamin D pathway activation and innate immune responses of mammary immune cells. Individual breast quarters of 5 lactating cows were treated with placebo control, 100 μg 25(OH)D₃, 5 μg LPS or a combination of 100 μg 25(OH)D₃and 5 μg of LPS. Milk somatic cells were evaluated for the percentage of neutrophil and macrophage populations and the expression of genes related to vitamin D metabolism and innate immunity. Data from samples collected 4 to 12 hours after exposure were analyzed for the main effects of LPS and 25(OH)D₃treatments, treatment interactions and simple effects of 25(OH)D₃therapy. Data from samples collected at the time of exposure were used as covariates. The percentages of neutrophils in milk 8 hours after exposure were 58 ± 10, 82 ± 11, 89 ± 10 and 63 ± 10% of the total number of cells in milk from control, 25(OH)D₃, LPS i LPS plus 25(OH)D₃glands, so that the interaction of LPS and 25(OH)D₃was significant. Gene expression of vitamin D 1α-hydroxylase (CYP27B1) and vitamin D receptor was up-regulated by LPS treatment in milk total cells, macrophages and neutrophils. In addition, the expression of the vitamin D 24-hydroxylase (CYP24A1) gene in somatic breast cells is up-regulated by 25(OH)D₃and LPS treatments. Inducible nitric oxide synthase (iNOS), chemokine (C-C motif) ligand 5 (CCL5), β-defensin 3 (DEFB3), DEFB7 and DEFB10 genes were upregulated by LPS treatment in total cells and milk neutrophils. The expression of iNOS in somatic breast cells was mainly influenced by the interaction between LPS and 25(OH)D₃, so that 25(OH)D₃tended to increase iNOS in the absence of LPS, but not in the presence of LPS. In addition, CCL5 expression in macrophages was reduced by 25(OH)D₃. In conclusion, intramammary endotoxin challenge activates the vitamin D pathway in mammary macrophages and neutrophils, and intramammary 25(OH)D₃treatment changes the percentage of neutrophils and the expression of immune genes in somatic milk cells.

Antibiotics have traditionally been included in animal feeds at sub-therapeutic levels to promote growth and prevent disease. However, recent links between antibiotics in animal feed and the emergence of antibiotic-resistant pathogens led to a ban on all antibiotics in livestock farming by the European Union in January 2006 and the removal of medically important antibiotics from animal feed in January 2006 in the United States. 2017. there is an urgent need for alternative antibiotics that can maintain animal health and productivity without causing antimicrobial resistance. Host defense peptides (HDPs) are a critical component of the innate immune system of animals with direct antimicrobial and immunomodulatory effects. While dietary supplementation of recombinant or synthetic HDP appears to be effective in maintaining animal performance and alleviating clinical signs in the context of disease, nutritional modulation of host endogenous defense peptide synthesis has emerged as a cost-effective, alternative approach to antibiotics for disease control and prevention. Several classes of small molecule compounds have been found that can promote HDP synthesis. Among the most effective compounds are butyrate and vitamin D. In addition, butyrate and vitamin D interact with each other in enhancing HDP synthesis. This review will focus on the regulation of HDP synthesis by butyrate and vitamin D in humans, chickens, pigs and cattle and argue for the potential application of HDP-inducing compounds in antibiotic-free livestock farming.

The Journal of Steroid Biochemistry and Molecular Biology, svezak 173, 2017., str. 139-147

Vitamin D has so far failed to live up to its early promise as an antineoplastic agent, despite compelling in vitro data. With the aim of effectively introducing vitamin D or its derivatives (VDD) into the clinic, we developed a two-pronged approach. First, by adding vegetable carnosic acid (CA) to the vitamin D2 derivative doxercalciferol, we increased its differentiation ability without increasing its hypercalcemic properties. Second, we added these two agents together to AML cells already treated with cytarabine (AraC), a standard drug for the treatment of patients with AML. We now report that BRAF, part of the MAPK signaling pathway, is required for optimally enhanced cell death in this system and acts upstream of BIM, a regulator of the caspase cascade leading to cell death by apoptosis. It is proposed to test this therapeutic regimen in a clinical trial.

The Journal of Steroid Biochemistry and Molecular Biology, svezak 173, 2017., str. 75-78

We previously found that 2α-[2-(tetrazol-2-yl)ethyl]-1α,25-dihydroxyvitamin D₃(AH-1) showed greater transactivation activity of the osteocalcin promoter in human osteosarcoma cells (HOS) and greater therapeutic effect in ovariectomized rats (OVX) to increase bone mineral density than that of 1α,25(OH)₂D₃without hypercalcemic side effects in vivo. Although CYP24A1 catalyzes multi-step oxidations to the side chain of the CD ring of active vitamin D₃[1α,25(OH)₂D₃] tended to halt the CYP24A1-dependent metabolism of AH-1 at the first hydroxylation step at the C24 position of AH-1. Interestingly, the metabolite 24-hydroxy-AH-1 [24(OH)AH-1] showed strong VDR binding affinity, and the new chiral center of position 24 could be the 24R configuration compared to the process of natural 1α , 25(OH)₂D₃catabolism. This time (24R)-2α-[2-(tetrazol-2-yl)ethyl]-1α,24,25-trihydroxyvitamin D₃[(24R-OH)AH-1] was synthesized as a candidate major metabolite of AH-1 using a Trost Pd-mediated coupling reaction between A ring and CD ring precursors to identify the metabolite and determine its biological activity. to evaluate. We confirmed by HPLC analysis that the major metabolite of AH-1 is dependent on CYP24A1 (24R-OH)AH-1.

The Journal of Steroid Biochemistry and Molecular Biology, svezak 173, 2017., str. 69-74

Gemini analogues of calcitriol, characterized by the second chain extension of the C21 methyl group of calcitriol, act as vitamin D receptor (VDR) agonists. This second Gemini side chain is placed in a new pocket in the VDR created by a structural rearrangement of the protein core. The resulting conformational change preserves the active state of the receptor and provides the Gemini compounds with biological activities that exceed those of calcitriol. Of particular interest are the anticancer properties of Gemini, and in this study we demonstrate the antiproliferative and tumor-reducing properties of BXL0124 and BXL0097, which differ only in the presence or absence of the methylene group on the A ring. BXL0124 acts as a more potent VDR agonist than its 19-nor counterpart by activating VDR-mediated transcription at lower concentrations. Similarly, BXL0124 is more active than BXL0097 in inhibiting breast cancer cell growth and reducing tumor volume. Structural comparisons of BXL0097 and BXL0124, as their VDR complexes, explain the increased activity of the latter.

The Journal of Steroid Biochemistry and Molecular Biology, svezak 173, 2017., str. 79-82

According to the binding mode of 14-epi-1a,25-dihydroxy-19-nortachysterol in the ligand-binding domain of the human vitamin D receptor (hVDR), i.e. 5,6- and 7,8-s-trans configuration represented by X-ray -co-crystallographic analysis, 7,8-cis-locked 1α,25(OH)₂D₃analogs were synthesized. This article presents the synthesis and biological activity of 2α- and 2β-(3-hydroxypropyl)-7,8-cis-14-epi-1α,25-dihydroxy-19-norvitamin D.₃have been reported. A-ring and CD-ring precursors for the Julia-Kociensky coupling reaction to generate the diene system of the target molecules were prepared using our original methods. hVDR binding affinity and osteocalcin promoter transactivation activity of the novel 7,8-cis-14-epi-vitamin D₃analogs were evaluated. Interestingly, the 2β-substituted 7,8-cis analog was a better binding agent for hVDR than its 2α-isomeric counterpart.

The Journal of Steroid Biochemistry and Molecular Biology, svezak 173, 2017., str. 83-85

As part of our program to search for vitamin D analogues with selective biological properties, such as low or negligible calcemic activity, we here describe an efficient and versatile synthetic approach to 1α,25-dihydroxyvitamin D derivatives₂with homologous side chains and substitution at C24 for biological evaluation.

The Journal of Steroid Biochemistry and Molecular Biology, svezak 173, 2017., str. 64-68

Several vitamin D 19-nor analogs, which lack the methylene moiety at C19, show significant vitamin D-related biological activities (VD), but generally show a reduced side effect of calcemia compared to VD alone. Among them, paricalcitol is already used clinically for the treatment and prevention of secondary hyperparathyroidism associated with chronic renal failure. Therefore, significant synthetic efforts are focused on 19-nor VD analogs, with a particular emphasis on A-ring synthons suitable for use in convergent synthetic strategies based on the coupling of CD-ring synthons and A-ring synthons. synthons. For example, we recently developed a new synthetic route to A-ring synthons from linear dienes based on a ring-closing olefin metathesis strategy. Here we review recent synthetic approaches to A-ring synthons for 19-nor VD derivatives.