Short communication: Vitamin D status and responses in dairy cows naturally infected with Mycobacterium avium ssp. paratuberculosis.

Serum samples were obtained from Holstein dairy control cows and cows naturally infected with Mycobacterium avium ssp. paratuberculosis (MAP) to evaluate the effects of disease status on serum 25-hydroxyvitamin D3 (25OHD3) levels. Disease status was stratified for infected cows into asymptomatic, subclinical infection (n = 25), and cows demonstrating clinical signs (n = 20), along with noninfected control (n = 12) cows for comparison. In addition, portions of the ileocecal valve were taken from a subsample of cows (n = 5 per treatment group) at necropsy and processed for RNA sequencing gene transcription studies. Genes associated with vitamin D metabolism were queried to determine any association between infection and gene expression. Serum 25OHD3 levels were significantly lower in cows in the clinical stage of disease compared with either cows in the subclinical stage and noninfected control cows. Differential expression for genes associated with the vitamin D pathway such as CYP27A1, CYP27B1, vitamin D-binding protein (DBP), and IFNG was dependent upon infection status. An upregulation of CYP27A1 was noted for cows in subclinical status, whereas CYP27B1 expression was enhanced for clinical cows. Increased expression of vitamin D-binding protein was observed for infected cattle, regardless of infection status. In summary, decreases in circulating 25OHD3 for animals with clinical disease may suggest that these cows have reduced innate immune responses, thereby influencing the ability of animals to fight infection.

Symposium review: Omics in dairy and animal science-Promise, potential, and pitfalls.

Sequencing the first genome took 15 yr and $3 billion to complete. Currently, a genome can be sequenced in a day for a few thousand dollars. Comparing the relative abundance of nearly every mRNA transcript and small RNAs from cells and tissues from different experimental conditions has become so easy that it can take longer to transfer the data between computers than to perform the experiment. Nucleotide sequencing techniques have become so sensitive that the greatest concern is not detecting a gene or transcript but rather, falsely identifying one. Better genome sequencing has led to more complete transcriptomic and proteomic databases and, combined with more sensitive instrumentation and separation techniques, is bringing us closer to detecting complete transcriptomes and proteomes. The promise of these powerful omics techniques is to lead us to new and unexpected connections between molecular processes in the context of animal health. This promise cannot be achieved without hypothesis-driven research that connects omics data with animal health experiments. Any researcher who wishes to invest the time and resources in omics experiments should be aware of the common pitfalls and limitations of these techniques so they can avoid these issues and maximize the use of these research tools. Several important questions must be asked: What is the quality of the databases and how they are annotated? Are the annotations based on experimental results or computational predictions? What assumptions are made by the analysis algorithms, and how will this affect the result? Finally, how can the research community use the vast amount of data being generated by omics experiments in ways to achieve the goals of better animal health and production (which is the promise of omics technologies)? Until the observations shown in omics data sets are used to achieve the goals of better animal health and production, the potential of omics technology will not be fully realized.

Differential phenotype of immune cells in blood and milk following pegylated granulocyte colony-stimulating factor therapy during a chronic Staphylococcus aureus infection in lactating Holsteins.

Neutrophils are principal host innate immune cell responders to mastitis infections. Thus, therapies have been developed that target neutrophil expansion. This includes the neutrophil-stimulating cytokine granulocyte colony-stimulating factor (gCSF). Pegylated gCSF (PEG-gCSF; Imrestor, Elanco Animal Health, Greenfield, IN) has been shown to reduce the natural incidence of mastitis in periparturient cows in commercial settings and reduce severity of disease against experimental mastitis challenge. Pegylated gCSF stimulates neutrophil expansion but also induces changes in monocyte and lymphocyte circulating numbers, surface protein expression changes, or both. We hypothesized that PEG-gCSF modulates surface expression of monocytes and neutrophils and facilitates their migration to the mammary gland. We challenged 8 mid-lactation Holsteins with approximately 150 cfu of Staphylococcus aureus (Newbould 305) in a single quarter via intramammary infusion. All animals developed chronic infections as assessed by bacteria counts and somatic cell counts (SCC). Ten to 16 wk postchallenge, 4 of the animals were treated with 2 subcutaneous injections of PEG-gCSF 7 d apart. Complete blood counts, SCC, bacterial counts, milk yield, feed intake, neutrophils extracellular trap analysis, and flow cytometric analyses of milk and blood samples were performed at indicated time points for 14 d after the first PEG-gCSF injection. The PEG-gCSF-treated cows had significantly increased numbers of blood neutrophils and lymphocytes compared with control cows. Flow cytometric analyses revealed increased surface expression of myeloperoxidase (MPO) on neutrophils and macrophages in milk but not in blood of treated cows. Neutrophils isolated from blood of PEG-gCSF-treated cows had decreased surface expression of CD62L (L-selectin) in blood, consistent with cell activation. Surprisingly, CD62L cell surface expression was increased on neutrophils and macrophages sourced from milk from treated animals compared with cells isolated from controls. The PEG-gCSF-treated cows did not clear the S. aureus infection, nor did they significantly differ in SCC from controls. These findings provide evidence that PEG-gCSF therapy modifies cell surface expression of neutrophils and monocytes. However, although surface MPO+ cells accumulate in the mammary gland, the lack of bacterial control from these milk-derived cells suggests an incomplete role for PEG-gCSF treatment against chronic S. aureus infection and possibly chronic mammary infections in general.

The effect of pegylated granulocyte colony-stimulating factor treatment prior to experimental mastitis in lactating Holsteins.

Neutrophils are the first-acting and most prominent cellular defense against mastitis-causing pathogens. This makes neutrophil activation and expansion obvious candidates for targeted therapeutics. The granulocyte colony-stimulating factor (G-CSF) cytokine stimulates the bone marrow to produce granulocytes and stem cells and release them into the bloodstream, which results in neutrophilia as well as increasing the presence of other progenitor cells in the bloodstream. A pegylated form of G-CSF (PEG-gCSF) has been shown to significantly decrease naturally occurring mastitis rates in cows postpartum. The use of PEG-gCSF had not been evaluated in response to an experimental mastitis challenge. In an effort to examine the effect and mechanism of PEG-gCSF treatment, we challenged 11 mid-lactation Holsteins with ∼400 cfu Escherichia coli P4 by intramammary infusion. Five cows received 2 PEG-gCSF injections, one at 14 d and the other at 7 d before disease challenge, and 6 cows remained untreated. To evaluate the response of cows to the PEG-gCSF treatment, we measured complete blood counts, somatic cell counts, bacterial counts, milk yield, and feed intake data. The PEG-gCSF-treated cows had significantly increased circulating levels of neutrophils and lymphocytes after each PEG-gCSF injection, as well as following mastitis challenge. The PEG-gCSF-treated cows had significantly lower bacterial counts and lower milk BSA levels at the peak of infection. In addition, control cows had significant decreases in milk yield postinfection and significantly reduced feed intake postinfection compared with PEG-gCSF-treated cows. Collectively, PEG-gCSF treatment resulted in reduced disease severity when administered before a bacterial challenge. Mechanistically, we show that G-CSF treatment increases cell surface expression of an E-selectin ligand before infection on neutrophils and monocytes found in the blood. These cells quickly disappear from the blood shortly after infection, suggesting a mechanism for the reduced mastitis severity by priming immune cells for quick targeting to the site of infection.

Effects of fractionated colostrum replacer and vitamins A, D, and E on haptoglobin and clinical health in neonatal Holstein calves challenged with Mycobacterium avium ssp. paratuberculosis.

Thirty Holstein calves were obtained from 2 dairy farms in central Iowa at birth and randomly assigned to 1 of 6 treatment groups: (1) colostrum deprived (CD), no vitamins; (2) colostrum replacer (CR), no vitamins; (3) CR, vitamin A; (4) CR, vitamin D3; (5) CR, vitamin E; and (6) CR, vitamins A, D3, E, with 5 calves per treatment in a 14-d study. Calves were fed pasteurized whole milk (CD) or fractionated colostrum replacer (CR) at birth (d 0) and injected with vitamins according to treatment group. From d 1 through d 14 of the study, all calves were fed pasteurized whole milk (PWM) supplemented with vitamins as assigned. All calves were inoculated with Mycobacterium avium ssp. paratuberculosis on d 1 and 3 of age. Calves fed CR acquired IgG1 and haptoglobin in serum within 24 h of birth, whereas CD calves did not. The CR-fed calves were 2.5 times less likely to develop scours, and CR calves supplemented with vitamins D3 and E also demonstrated a decreased incidence of scours. Serum vitamin levels of A, D, and E increased within treatment group by d 7 and 14 of the study. Interestingly, synergistic effects of supplemental vitamins A, D3, and E on serum 25-(OH)-vitamin D were observed at d 7, resulting in higher levels than in calves administered vitamin D only. Further, vitamin D3 deficiency was observed in CD and CR calves fed a basal diet of pasteurized whole milk and no supplemental vitamins. Colonization of tissues with Mycobacterium avium ssp. paratuberculosis was negligible and was not affected by colostrum feeding or vitamin supplementation. Results demonstrated passive transfer of haptoglobin to neonatal calves, and potential health benefits of supplemental vitamins D3 and E to calves fed pasteurized whole milk.

Effect of feeding 25-hydroxyvitamin D3 with a negative cation-anion difference diet on calcium and vitamin D status of periparturient cows and their calves.

Holstein cows (>1 gestation) were fed 1 of 3 diets during the last 13 d of gestation (ranged from 22 to 7 d). The control diet (16 cows) was formulated to provide 18,000 IU/d of vitamin D3 and had a dietary cation-anion difference (DCAD) of 165mEq/kg (DCAD=Na + K - Cl - S). The second diet (DCAD + D) provided the same amount of vitamin D3 but had a DCAD of -139mEq/kg (17 cows). The third diet (DCAD + 25D) had no supplemental vitamin D3 but provided 6mg/d of 25-(OH) vitamin D3 [25-(OH)D3] with a DCAD of -138mEq/kg (20 cows). Diets were fed until parturition and then all cows were fed a common lactation diet that contained vitamin D3. Negative DCAD diets reduced urine pH, with the greatest decrease occurring with the DCAD + D treatment. Urinary Ca excretion was greatest for cows fed DCAD + 25D followed by cows fed DCAD + D. Urinary pH was negatively correlated with urinary excretion of Ca for cows fed DCAD + D. No such correlation was observed with the DCAD + 25D treatment because substantial excretion of urinary Ca occurred at moderate urinary pH values for that treatment. Cows fed DCAD + 25D had greater serum concentrations of 25-(OH)D3 than other treatments from 5 d after supplementation started through 7 d in milk. Concentrations of 1,25-(OH)2D3 in serum were greatest in DCAD + 25D cows starting at 2 d before calving and continued through 7 d in milk. Serum Ca concentrations 5 d before calving were greatest for cows fed DCAD + 25D, but at other time points before and after parturition treatment did not affect serum Ca. Incidence of clinical hypocalcemia was not statistically different between treatments, but cows fed DCAD + 25 had the highest incidence rate (12.5, 0, and 20% for control, DCAD + D, and DCAD + 25D). Calves born from cows fed DCAD + 25D had greater concentrations of 25-(OH)D3 in serum at birth than calves from other treatments (before colostrum consumption), but concentrations were similar by 3 d of age. Concentrations of 25-(OH)D3 in colostrum and transition milk were increased by feeding DCAD + 25D, but by 28 d in milk treatment effects no longer existed. Overall, feeding 25-OH vitamin D with a negative DCAD diet increased vitamin D status of the cow and her newborn calf but had minimal effects on calcium status and did not have positive effects on the incidence of hypocalcemia.

Acute phase response elicited by experimental bovine diarrhea virus (BVDV) infection is associated with decreased vitamin D and E status of vitamin-replete preruminant calves.

Studies in young animals have shown an association between vitamin deficiencies and increased risk of infectious disease; however, there is a paucity of information regarding the effect of acute infection on the vitamin status of the vitamin-replete neonate. To characterize the effects of acute infection on vitamin D and E status of the neonate, 6 vitamin-replete preruminant Holstein bull calves were experimentally infected with bovine viral diarrhea virus (BVDV; strain BVDV2-1373). Six mock-inoculated calves served as controls. Sustained pyrexia, leukopenia, and asynchronous increases in serum haptoglobin and serum amyloid A characterized the response of calves to infection with BVDV. Infection was also associated with increased serum IFN-γ, IL-2, and IL-6 concentrations. During the last 8 d of the 14-d postinoculation period, serum 25-hydroxyvitamin D and α-tocopherol concentrations in infected calves decreased by 51 and 82%, respectively. The observed inverse association between vitamin D and E status and serum amyloid A in infected calves suggests that the infection-induced acute phase response contributed to the reduced vitamin status of these animals. Additional studies are necessary to determine if the negative effect of infection on status are unique to this specific infection model or is representative of preruminant calf's response to acute infection. Studies are also needed to characterize mechanisms underlying infection-related changes in vitamin D and E status and to determine whether additional vitamin D or E supplementation during an acute infection diminishes disease severity and duration in the young animal.

Modulation of the bovine innate immune response by production of 1alpha,25-dihydroxyvitamin D(3) in bovine monocytes.

In cattle, the kidney has been the only known site for production of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] from 25-hydroxyvitamin D(3) [25(OH)D(3)] by 1alpha-hydroxylase (1alpha-OHase). Based on human studies, it was hypothesized that bovine monocytes could produce 1,25(OH)(2)D(3) upon activation and 1,25(OH)(2)D(3) would regulate expression of vitamin D-responsive genes in monocytes. First, the effects of 1,25(OH)(2)D(3) on bovine monocytes isolated from peripheral blood were tested. Treatment of nonstimulated monocytes with 1,25(OH)(2)D(3) increased expression of the gene for the vitamin D 24-hydroxylase (24-OHase) enzyme by 51+/-13 fold, but 1,25(OH)(2)D(3) induction of 24-OHase expression was blocked by lipopolysaccharide (LPS) stimulation. In addition, 1,25(OH)(2)D(3) increased the gene expression of inducible nitric oxide synthase and the chemokine RANTES (regulated upon activation, normal T-cell expressed and secreted) in LPS-stimulated monocytes 69+/-13 and 40+/-12 fold, respectively. Next, the ability of bovine monocytes to express 1alpha-OHase and produce 1,25(OH)(2)D(3) was tested. Activation of monocytes with LPS, tripalmitoylated lipopeptide (Pam3CSK4), or peptidoglycan caused 43+/-9, 17+/-3, and 19+/-3 fold increases in 1alpha-OHase gene expression, respectively. Addition of 25(OH)D(3) to LPS-stimulated monocytes enhanced expression of inducible nitric oxide synthase and RANTES and nitric oxide production in a dose-dependent manner, giving evidence that activated monocytes convert 25(OH)D(3) to 1,25(OH)(2)D(3). In conclusion, bovine monocytes produce 1,25(OH)(2)D(3) in response to toll-like receptor signaling, and 1,25(OH)(2)D(3) production in monocytes increased the expression of genes involved in the innate immune system. Vitamin D status of cattle might be important for optimal innate immune function because 1,25(OH)(2)D(3) production in activated monocytes and subsequent upregulation of inducible nitric oxide synthase and RANTES expression was dependent on 25(OH)D(3) availability.

Short communication: the preruminant calf as a model for characterizing the effects of vitamin D status in the neonate.

The objective of this study was to evaluate the feasibility of using the preruminant dairy calf as a model for evaluating effects of vitamin D status in the neonate. Because the newborn calf can be sustained during the first weeks of life solely on a fluid diet having a defined composition, has documented nutritional requirements, and is minimally affected by repeated samplings of peripheral blood, it has the potential to serve as a model for characterizing nutrient-specific effects on the growth and health of the neonate. Colostrum-fed Holstein bull calves (n = 13) entered the trial at approximately 4 d of age. All calves were fed a custom-formulated milk replacer devoid of vitamin D. Plasma 25-hydroxyvitamin D(3) concentrations in all calves were determined on a regular basis beginning at d 0. Using this information, low- and high-status groups of calves were established by subcutaneous administration of 25-hydroxyvitamin D(3). To maintain targeted plasma 25-hydroxyvitamin D(3) concentrations in low (<30 ng/mL) and high (>60 ng/mL) vitamin D-status calves, low-status calves (n = 6) received a total of 8,600 IU (2,225 IU/wk) of vitamin D during the experimental period and high-status calves (n = 7) received 54,000 IU (13,500 IU/wk). Concentrations of 25-hydroxyvitamin D(3) in low-status calves averaged 27 ng/mL, compared with 78 ng/mL in high-status calves, and were less at all sampling times from d 7 to d 28. Concentrations of 1,25-dihydroxyvitamin D(3) and 25-hydroxyvitamin D(3) were not correlated. Calcium, magnesium, and phosphorous concentrations were unaffected by 25-hydroxyvitamin D(3) administration; however, plasma calcium and 1,25-dihydroxyvitamin D(3) concentrations were correlated. Calcium and magnesium concentrations decreased with age but remained within normal ranges for dairy cattle. These results indicate that it is possible to predictably control vitamin D status over a 28-d period and suggest that the preruminant calf might be useful as a model for studying effects of vitamin D on growth, development, and immune function in the neonate.

Developmental changes in the milk fat globule membrane proteome during the transition from colostrum to milk.

Shotgun proteomics, using amine-reactive isobaric tags (iTRAQ), was used to quantify protein changes in milk fat globule membranes (MFGM) that were isolated from d 1 colostrum and compared with MFGM from d 7 milk. Eight Holstein cows were randomly assigned to 2 groups of 4 cow sample pools for a simple replication of this proteomic analysis using iTRAQ. The iTRAQ labeled peptides from the experiment sample pools were fractionated by strong cation exchange chromatography followed by further fractionation on a microcapillary high performance liquid chromatograph connected to a nanospray-tandem mass spectrometer. Data analysis identified 138 bovine proteins in the MFGM with 26 proteins upregulated and 19 proteins downregulated in d 7 MFGM compared with colostrum MFGM. Mucin 1 and 15 were upregulated greater than 7-fold in MFGM from d 7 milk compared with colostrum MFGM. The tripartite complex of proteins of adipophilin, butyrophilin, and xanthine dehydrogenase were individually upregulated in d 7 MFGM 3.4-, 3.2-, and 2.6-fold, respectively, compared with colostrum MFGM. Additional proteins associated with various aspects of lipid transport synthesis and secretion such as acyl-CoA synthetase, lanosterol synthase, lysophosphatidic acid acyltransferase, and fatty acid binding protein were upregulated 2.6- to 5.1-fold in d 7 MFGM compared with colostrum MFGM. In contrast, apolipoproteins A1, C-III, E, and A-IV were downregulated 2.6- to 4.3-fold in d 7 MFGM compared with colostrum MFGM. These data demonstrate that quantitative shotgun proteomics has great potential to provide new insights into mammary development.

Novel role for IL-22 in protection during chronic Mycobacterium tuberculosis HN878 infection.

Approximately 2 billion people are infected with Mycobacterium tuberculosis (Mtb), resulting in 1.4 million deaths every year. Among Mtb-infected individuals, clinical isolates belonging to the W-Beijing lineage are increasingly prevalent, associated with drug resistance, and cause severe disease immunopathology in animal models. Therefore, it is exceedingly important to identify the immune mechanisms that mediate protection against rapidly emerging Mtb strains, such as W-Beijing lineage. IL-22 is a member of the IL-10 family of cytokines with both protective and pathological functions at mucosal surfaces. Thus far, collective data show that IL-22 deficient mice are not more susceptible to aerosolized infection with less virulent Mtb strains. Thus, in this study we addressed the functional role for the IL-22 pathway in immunity to emerging Mtb isolates, using W-Beijing lineage member, Mtb HN878 as a prototype. We show that Mtb HN878 stimulates IL-22 production in TLR2 dependent manner and IL-22 mediates protective immunity during chronic stages of Mtb HN878 infection in mice. Interestingly, IL-22-dependent pathways in both epithelial cells and macrophages mediate protective mechanisms for Mtb HN878 control. Thus, our results project a new protective role for IL-22 in emerging Mtb infections.

Parturition and hypocalcemia blunts calcium signals in immune cells of dairy cattle.

The stress of parturition in the dairy cow is associated with increased susceptibility to infectious disease. During the periparturient period the demands for calcium are increased; these increased demands for calcium can result in subclinical or clinical hypocalcemia. Periparturient cows also experience significant immune suppression. Because intracellular calcium signaling is a key early feature in immune cell activation, we have hypothesized that the increased demand for calcium in periparturient cows may adversely affect intracellular calcium stores of immune cells. This reduction in intracellular calcium stores in immune cells could blunt intracellular calcium release following an activating stimulus, contributing to the immune suppression seen in these animals. To test this hypothesis, peripheral mononuclear cells were obtained from 27 multiparous dairy cows spanning a period of 2 wk before and 2 wk after parturition. Following activation of these cells by anti-CD3 antibodies plus secondary antibodies, intracellular calcium release from intracellular stores was measured. The intracellular calcium released in response to the activation signal declined as calcium demand for lactation became more intense and recovered as plasma calcium normalized. Intracellular calcium stores in peripheral mononuclear cells, estimated by pretreating cells with pervanadate and ionomycin, significantly decreased at parturition and returned to normal levels as the cows' blood calcium returned to normal levels. Hypocalcemia, which is common in periparturient dairy cows, is associated with decreased intracellular calcium stores in peripheral mononuclear cells. Our data suggest that this is the cause of a blunted intracellular calcium release response to an immune cell activation signal. It is concluded that intracellular Ca stores decrease in peripheral blood mononuclear cells (PBMC) before parturition and development of hypocalcemia. This suggests that systemic calcium stress precedes measurable hypocalcemia, particularly in cows that will develop milk fever. Therefore, PBMC intracellular Ca stores are a more sensitive measure of calcium stresses in transition cow. This decrease in PBMC intracellular Ca stores before parturition and the development of hypocalcemia contributes to periparturient immune suppression.

Seasonal variation in vitamin D status of beef cattle reared in the central United States.

The objective was to retrospectively measure seasonal sunlight-associated variation in serum concentrations of 25-hydroxyvitamin D (25OHD) in beef cattle. The concentration of 25OHD was measured in crossbred animals born from March to May in 2011 and 2012. Vitamin D status 2 to 3 mo after birth (period 1) was only available for 2012 calves and was measured in June 2012. Period 1 animals had serum 25OHD concentrations of 26.3 ± 1.5 ng/mL. The 25OHD concentrations for late summer (period 2) were 46.6 ± 1.4 and 51.0 ± 1.5 ng/mL for 2011 and 2012, respectively. Serum concentration of 25OHD in early fall (period 3) were 63.8 ± 1.4 and 55.2 ± 1.5 ng/mL for calves in 2011 and 2012, respectively. Values observed for both late summer and early fall indicated vitamin D sufficiency (P < 0.001) compared with period 1. With diminishing exposure to ultraviolet B and consuming ∼800 IU or 1800 IU (2011 and 2012, respectively) of supplemental vitamin D, the calves' midwinter (period 4) 25OHD concentrations fell to 15.2 ± 1.6 and 16.7 ± 1.5 ng/mL for 2011 and 2012, respectively, after 4 to 5 mo on a finishing diet (P < 0.0001). This is considered vitamin D insufficiency in most species. Results indicate that calves are marginally sufficient to insufficient for vitamin D based on serum 25OHD concentrations soon after birth and during winter. Some individual animals would be classified vitamin D deficient. In the absence of sufficient UVB exposure, the dietary vitamin D requirements for rapidly growing beef cattle may need to be increased.

Differential effects of 20-epi vitamin D analogs on the vitamin D receptor homodimer.

Vitamin D analogs have received increased attention because of their possible therapeutic benefits in treating osteoporosis and various proliferative disorders. Several analogs were examined for their effects on DNA binding of the vitamin D receptor (VDR) homodimer complex with the murine osteopontin vitamin D response element. All of the tested analogs increased complex binding by recombinant human VDR in the electrophoretic mobility shift assay and notable differences in mobility of these complexes were observed. A panel of C-terminal anti-VDR antisera were screened for their ability to interact with analog-bound VDR homodimer complexes or as a heterodimer complex with recombinant human retinoid X receptor alpha (rhRXR alpha). Like calcitriol, analog-bound heterodimer complexes were largely resistant to interaction with these antisera; however, striking differences were observed with the various antisera in an analogous homodimer binding experiment. KH1060 and CB1093, analogs with 20-epi conformations, produced homodimer complexes that were 3- to 6-fold more resistant to supershifting with Ab180 compared with the hormone or EB1089. Chymotrypsin digestion in combination with Western blotting using a C-terminal anti-VDR antiserum revealed similar digestion patterns for all ligands. However, KH1060- and CB1093-bound VDR complexes were more resistant to digestion than either calcitriol or EB1089. Finally, the ability of these compounds to yield stable homodimer complexes was assessed by challenging preformed homodimer with the exogenous addition of rhRXR alpha extracts. Although new heterodimer complexes appeared in a time-dependent fashion, the preformed homodimer complexes exhibited stable binding throughout the time course of the experiment. The results indicate that VDR homodimers are targets of vitamin D analogs with differential effects on C-terminal protein conformation that may partially explain the varied biological responses of these compounds.

Parathyroid hormone down-regulates 1,25-dihydroxyvitamin D receptors (VDR) and VDR messenger ribonucleic acid in vitro and blocks homologous up-regulation of VDR in vivo.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3) is a known up-regulator of 1,25(OH)2D3 receptor (VDR) both in vitro and in vivo. However, a 5- to 10-fold increase in plasma 1,25(OH)2D3 induced by dietary calcium deficiency does not result in up-regulation of intestinal VDR, and kidney VDR is down-regulated. Under certain physiological stresses, an increase in plasma PTH precedes increased plasma 1,25(OH)2D3. Therefore, the present study examined the effect of PTH on VDR regulation in vitro in ROS 17/2.8 cells and in vivo in male Holtzman rats. Treatment of ROS cells with PTH (0-5 nM) resulted in a dose and time-dependent decline in VDR from 95 +/- 9 to 35 +/- 5 fmol/mg protein at 18 h of exposure. The ED50 for PTH was 1 nM. This decline in VDR protein was attended by a 50% decline in VDR messenger RNA (mRNA). The PTH-mediated down-regulation of VDR occurred without affecting the affinity of VDR for 1,25(OH)2D3 as determined by Scatchard analysis. Also, the effect of PTH on VDR regulation was specific since cell glucocorticoid receptor concentration was not affected by PTH treatment. In accompanying experiments, 1,25(OH)2[3H]D3 treatment of ROS cells was shown to result in a 3- to 4-fold increased expression of VDR and VDR mRNA. The simultaneous addition of PTH and 1,25(OH)2[3H]D3 resulted in inhibition of the 1,25(OH)2[3H]D3-mediated up-regulation of VDR and VDR mRNA. Similarly, PTH also inhibited heterologous up-regulation of VDR and VDR mRNA induced by retinoic acid. In in vivo experiments, rats infused for 5 days with 1,25(OH)2D3 (1.5 ng/h) increased their expression of intestinal VDR, kidney VDR, and kidney 24-hydroxylase by 31, 336, and 4000%, respectively. Coinfusion of PTH (1.8 IU/h) along with 1,25(OH)2D3 completely inhibited the 1,25(OH)2D3-mediated increases in intestinal VDR and kidney 24-hydroxylase and reduced the 1,25(OH)2D3-mediated up-regulation of kidney VDR by more than half. These data suggest that PTH is a potent down-regulator of VDR and that PTH and 1,25(OH)2D3 have opposing effects on the expression of certain genes.

Advancing age results in reduction of intestinal and bone 1,25-dihydroxyvitamin D receptor.

Target tissue 1,25-dihydroxyvitamin D [1,25-(OH)2D] receptor was monitored in adult (15- to 18-month-old) and young (4- to 5-week-old) male Holtzman rats. The concentration of unoccupied receptor (femtomoles per mg protein) was significantly higher in the intestine (666 +/- 19 vs. 162 +/- 43) and bone (61 +/- 3.7 vs. 18 +/- 2.5) of young rats compared to that in adults. The dissociation constant (Kd) of the intestinal receptor, however, remained very similar at both ages (young, 0.24 nM; adult, 0.53 nM). A similar age-related decline in unoccupied intestinal receptor was also observed in Fischer 344 rats and cows. Infusion of young and adult Holtzman rats with about 72 ng/kg BW 1,25-(OH)2D3 resulted in similar changes in the concentrations of plasma 1,25-(OH)2D3 (150-160 pg/ml) in both age groups. The 1,25-(OH)2D3 infusions also resulted in significant up-regulation of unoccupied intestinal receptor (femtomoles per mg protein) from 512 +/- 27 to 780 +/- 61 in the young rats and 68 +/- 9.4 to 194 +/- 15 in the adult rats. Receptor up-regulation by 1,25-(OH)2D3 (change from control) was significantly higher (P less than 0.05) in young rats (268 +/- 51 fmol/mg protein) than in adults (125 +/- 8.2 fmol/mg protein). These data suggest that the differences in receptor number in young and adult rats may be responsible for functional changes in target tissue response to 1,25-(OH)2D3 associated with advancing age.

Pregnancy and lactation increase vitamin D-dependent intestinal membrane calcium adenosine triphosphatase and calcium binding protein messenger ribonucleic acid expression.

The calcium demands of pregnancy and lactation are known to up-regulate intestinal calcium absorption. Intestinal epithelial cells contain calcium ATPases and calcium binding proteins, which are believed to play important roles in intestinal calcium transport. However, the possible role of these two proteins in the up-regulation of intestinal calcium absorption observed in pregnancy and lactation is unknown. In this study, intestinal calcium ATPase (PMCA1), calcium binding protein (9K) (CaBP-9K), and vitamin D receptor (VDR) messenger RNA (mRNA) levels were determined by Northern analysis at different stages of pregnancy and early lactation in rats. Intestinal calcium ATPase and calcium binding protein mRNA levels did not differ significantly among nonpregnant rats and rats pregnant for 7 or 14 days. However, at 21 days gestation both calcium ATPase and calcium binding protein mRNA levels increased 2- to 3-fold. Calcium ATPase and calcium binding protein mRNA remained elevated at 7 days of lactation. Plasma 1,25-dihydroxyvitamin D3 (1,25-D3) concentration exhibited a similar pattern, rising markedly at 21 days gestation and remaining elevated in lactation. VDR mRNA levels did not change during the entire experiment. However, intestinal VDR content increased 2-fold in late pregnancy and lactation. These data suggest that transcription of calcium absorption factors is increased in late gestation and early lactation, perhaps mediated by increased plasma 1,25-dihydroxyvitamin D3 concentrations, and that the effects of gestation and lactation on VDR concentrations are probably posttranscriptional.

Parathyroid hormone-related peptide content of bovine milk and calf blood assessed by radioimmunoassay and bioassay.

PTH-related peptide (PTH-rP) has recently been discovered to exist in high concentrations in milk. The development of a commercial RIA for PTH-rP has allowed us to extend these studies. We measured the PTH-rP content of milk from 42 Jersey cows from a single farm in various stages of lactation. Colostrum (first milk) contained 56 +/- 12 ng/ml immunoreactive PTH-rP (iPTH-rP). The iPTH-rP contents of milk 1, 2, 3, 5, 7, and 9 months into lactation were 77 +/- 19, 59 +/- 14, 57 +/- 10, 106 +/- 11, 119 +/- 16, and 168 +/- 17 ng/ml, respectively. Plasma was obtained from 7 Jersey calves at birth and at intervals after the ingestion of colostrum. No iPTH-rP was detected in the plasma at birth. Two hours after the ingestion of colostrum, the iPTH-rP content of plasma was 81 +/- 25 pg/ml. The plasma iPTH-rP concentration continued to increase to 384 +/- 84 pg/ml at 7 h and peaked at 444 +/- 84 pg/ml 12 h after birth. Two calves were sampled through the 60th hour after birth, at which time plasma iPTH-rP was 483 +/- 36 pg/ml. The biological activity of the PTH-rP in milk and plasma was assessed by its ability to stimulate cAMP accumulation in ROS 17/2.8 cells. The specificity of this response was determined by the ability of antiserum to PTH-rP to block the activity. The biological activity of the milk samples was between 31-95% of the activity suggested by immunoassay. Biologically active PTH-rP could not be detected in any of the calf plasma samples. These results confirm the presence of biologically active PTH-rP in milk and suggest that the iPTH-rP is capable of being absorbed. However, our results indicate that the biological activity of the PTH-rP is nearly completely absent once in the systemic circulation.

Recurring hypocalcemia of bovine parturient paresis is associated with failure to produce 1,25-dihydroxyvitamin D.

Parturient paresis (milk fever) is a hypocalcemic disorder caused by the onset of lactation in the dairy cow. In most cows a complete recovery follows a single iv calcium treatment to correct the acute hypocalcemia. However, about 20% of cows treated for parturient paresis experience recurring episodes of hypocalcemia (relapses) requiring further treatment. Analysis of plasma from 8 nonrelapsing parturient paretic and 11 relapsing parturient paretic cows revealed differences in plasma 1,25-dihydroxyvitamin D [1,25-(OH)2D] concentrations before and during the development of hypocalcemia. In nonrelapsing cows, plasma 1,25-(OH)2D increased to 4- to 5-fold as plasma calcium concentrations declined during the first stage of parturient paresis. In relapsing cows, decreases in plasma calcium concentrations during the first stage of parturient paresis were accompanied by just a 2- to 2.5-fold increase in plasma 1,25-(OH)2D. Plasma 1,25-(OH)2D eventually increased 4- to 5-fold in the relapsing cows, but this response was delayed 24-48 h compared with the response in the nonrelapsing cows. Plasma PTH concentration profiles were similar in relapsing and nonrelapsing cows, suggesting that renal 25-hydroxyvitamin D 1 alpha-hydroxylase was temporarily refractory to stimulation by PTH in the relapsing cows. In both groups of cows recovery from parturient paresis began about 12-24 h after plasma 1,25-(OH)2D concentrations had increased 4- to 5-fold. These data imply that lack of production of 1,25-(OH)2D is an important factor in predisposing the cow to relapses of parturient paresis and is critical for recovery from the hypocalcemia associated with the onset of lactation.